Fig 1.
The MEP pathway of isoprenoid synthesis and how its inhibition induces persistence in C. trachomatis.
1-deoxy-D-xylulose 5-phosphate synthase (DXS) converts initial MEP pathway substrates pyruvate and glyceraldehyde 3-phosphate to 1-deoxy-D-xylulose 5-phosphate (DXP). Dxr converts DXP to MEP. Two additional steps occur generating 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP). MEcPP is converted to 4-hydroxy-3-methylbutenyl 1-diphosphate (HMBPP) by IspG. IspH converts HMBPP to the isoprenoid precursor molecules IPP/DMAPP. IPP/DMAPP are stereoisomers produced in a 5:1 ratio and are used to generate isoprenoids. In bacteria, the isoprenoid bactoprenol (purple rectangle), is critical for peptidoglycan synthesis. 1, Peptidoglycan precursor Lipid II is synthesized in the bacterial cytoplasm through a series of enzymatic reactions and is anchored to the inner leaflet of the inner membrane of the Gram negative bacterial cell envelope via bactoprenol; 2, The lipid II molecule is flipped into the periplasm; 3, Periplasmic lipid II is incorporated into the peptidoglycan ring and bactoprenol is recycled. We postulate that both FSM and iron deprivation can disrupt isoprenoid synthesis since IspG and IspH are iron-sulfur cluster-containing enzymes. In both cases, isoprenoid synthesis is halted, bactoprenol availability is reduced and peptidoglycan synthesis is prevented, resulting in persistence induction in Chlamydia.
Fig 2.
Comparison of the predicted structure of DxrCT and the crystal structure of DxrEC.
(A) Chemical structure (upper) and 3D structure (lower) of fosmidomycin (FSM). 3D FSM structure is in similar orientation to chemical structure, but with reactive oxygen groups labeled (“O” = oxygen; “OP” = oxygen-phosphate). (B) The superimposed view of the predicted crystal structure of DxrCT (orange) over the crystal structure of DxrEC (blue). (C) Enlarged view of the FSM binding pocket from the superimposed structures in panel B. The amino acid residues in the binding pocket of DxrEC (blue) that bind to FSM are conserved in DxrCT (labeled and highlighted in magenta). FSM is shown in green with OP2, OP3, O2, and O1 sites labeled to indicate orientation.
Fig 3.
Expression of DxrCT in trans supports growth of an E. coli dxr conditionally lethal mutant.
Five μL of an overnight culture of E. coli MG1655 Δdxr::kan pBAD24::dxrEC (ATM1471) or E. coli MG1655 Δdxr::kan pBAD24::dxrCT (ATM1470) grown in the presence of 0.2% arabinose were streaked for isolation on an LB agar plate containing 0.5% arabinose (A) or 0.5% glucose (B). Images are representative of three independent experiments performed in duplicate.
Table 1.
Escherichia coli strains and plasmids used in this study.
Fig 4.
Overexpression of DxrCT in E. coli supports growth and cellular morphology under FSM exposure.
Overnight cultures of E. coli MG1655 carrying the indicated plasmids were subcultured 1:100 in LB broth + 0.2% arabinose either without FSM, or with 25 μM FSM. OD600 (A) and CFU (B) were determined every two hours for 6 hours. Phase contrast microscopy (C) was performed every two hours from T2 to T6. Arrows indicate rounded cell morphology observed during FSM exposure. Scale bars represent 10 μm. OD600 and microscopy data (A and C) are representative of three independent experiments, each performed with duplicate cultures. For CFU data (B) each curve depicts a single replicate that is representative of two independent experiments preformed in duplicate. Error bars in panel A represent the average of duplicate cultures from a single experiment +/- SD.
Fig 5.
Chlamydial inclusion size and production of infectious EB are significantly inhibited by FSM exposure.
HeLa cells were infected with C. trachomatis at an MOI of 0.5 and harvested for analysis at 40 hpci. (A) Representative 400X magnification fluorescence microscopy images of chlamydial inclusions (green) stained with BioRad anti-chlamydial LPS Pathfinder stain and HeLa cell nuclei (blue) counter-stained with DAPI. Scale bars = 50 μm. Insert from 2 mM FSM concentration depicts zoomed-in view of ABs. Scale bars = 10 μm. (B) Percent infectivity was calculated by counting the number of inclusions/cell nuclei per field in 10 fields per coverslip in triplicate samples. (C) The area of 150 random inclusions per triplicate sample were measured using the spline contour tool in the Zen Blue Zeiss software package. (D) Production of infectious EBs was determined via chlamydial titer assays by subpassage. Significant (p ≤ 0.005) difference from untreated samples is indicated by an asterisk (*). Error bars indicate +/- SEM from triplicate samples and data are representative of three independent experiments.
Fig 6.
FSM induces persistence in Chlamydia-infected HeLa cell cultures.
At 40 hpci, cultures were washed once with complete media and either re-fed with media containing FSM (replete) or without FSM (recovered). Samples were incubated an additional 80 h and titers (A) were examined at 120 hpci. Asterisk (*) indicates significant difference from 120 h, 2 mM replete samples. Error bars indicate +/- SEM from triplicate samples and data are representative of three independent experiments. (B) Electron micrographs from untreated and 2 mM FSM-treated Chlamydia-infected HeLa cells 40 hpci. Yellow arrows and black arrows indicate normal EBs and RBs, respectively. AB indicates aberrant bodies. Scale bars = 2 μm. For AMP insert (lower right panel), scale bars = 500 μm.
Fig 7.
Structured illumination microscopy of D-amino acid dipeptide (DAAD) peptidoglycan labeling in FSM-treated C. trachomatis.
Structured illumination microscopy was conducted on untreated, FSM-treated, or AMP-treated C. trachomatis-infected HeLa cells. 1 mM EDA-DA and either 1 μg/mL AMP or 2.44 mM FSM were added at T0 and coverslips were fixed at 14 h (A) or 38 h (B) post chlamydial infection. Peptidoglycan labeling (represented by EDA—DA) is shown in green and the C. trachomatis major outer membrane protein (MOMP) is shown in red. Images are representative of ~20 inclusions viewed per condition. Scale bars = 1 μm.