Conservation of the glycogen metabolism pathway underlines a pivotal function of storage polysaccharides in Chlamydiae

The order Chlamydiales includes obligate intracellular pathogens capable of infecting mammals, fishes and amoeba. Unlike other intracellular bacteria for which intracellular adaptation led to the loss of glycogen metabolism pathway, all chlamydial families maintained the nucleotide-sugar dependent glycogen metabolism pathway i.e. the GlgC-pathway with the notable exception of both Criblamydiaceae and Waddliaceae families. Through detailed genome analysis and biochemical investigations, we have shown that genome rearrangement events have resulted in a defective GlgC-pathway and more importantly we have evidenced a distinct trehalose-dependent GlgE-pathway in both Criblamydiaceae and Waddliaceae families. Altogether, this study strongly indicates that the glycogen metabolism is retained in all Chlamydiales without exception, highlighting the pivotal function of storage polysaccharides, which has been underestimated to date. We propose that glycogen degradation is a mandatory process for fueling essential metabolic pathways that ensure the survival and virulence of extracellular forms i.e. elementary bodies of Chlamydiales.


Supplementary
: Organization of glg genes of GlgC pathway across Chlamydial genomes. Genes encoding for ADP-glucose pyrophosphorylase (glgC); glycogen synthase (glgA), glycogen branching enzyme (glgB), glycogen phosphorylase (glgP) and glycogen debranching enzyme were listed according to their organization on chlamydial genomes. With a notable exception for glgC and glgP genes, which are often separated by one or two genes, most of glg genes are encoded more than 10 kpb from each other.

Supplementary Table 2:
List of pairs of primers used for cloning the genes involved in glycogen metabolism pathway of E. lausannensis and W. chondrophila and for heterologous secretion assay. Underlined nucleotides represent the attB sites added to the amplified genes that allow the cloning into pDONR 221 vectors following the recommendation of Thermofisher (Gateway TM ). Figure 1: a Domain organization of fused protein GlgA-GlgB of E. lausannensis and W. chondrophila. Glycogen synthase domain (gray box) and branching enzyme domain (white box) are respectively located at the N-(Nt) and C-termini (Ct) respectively. The insertion of one-nucleotide in E. lausannensis sequence results in a frame shift and the appearance of truncated GlgA-GlgB protein. Regions I, II and III represent highly conserved sequences in the glycogen synthase GT5 family that includes amino acid residues involved in the catalytic site and nucleotide binding sites. b Proton-NMR analyses of glycogen from rabbit liver and maltoheptaose (10 mg.mL -1 ) + ADP-glucose (3 mM) incubated overnight at 30°C in the presence (c) or in the absence (d) of recombinant GlgA-GlgB fusion enzyme of Waddlia Chondrophila. After incubation, enzymatic reactions were boiled and purified through anion and cation exchange resins (DOWEX 1 X 8 and DOWEX 50 W X 8). Protons involved in α-1,4 linkages or α-1,6 linkages resonate at 5.4 and 4.95 ppm, respectively. Protons in α and β position on C1 (reducing end) generate signals at 5.23 and 4.65 ppm. The absence of signal at 4.95 ppm suggests that either signal corresponding to α-1,6 linkages is below threshold of detection (<1%) or GlgB domain is not active in the GlgA-GlgB of W. chondrophila. Figure 2: SDS-PAGE analyses of recombinant GlgE after affinity column purification and determination of optima pH and temperature of GlgE-EL. Mean and standard deviation of three independent experiments are plotted. a His-tag GlgE of E. lausannensis and b W. chondrophila were expressed in Rosetta TM E. coli strain. After induction at mid exponential growth with IPTG for GlgE-EL and culture in auto-inductible medium for GlgE-WC, the overnight cultures were harvested by centrifugation. Cell pellets were suspended in loading buffer containing 25 mM TRIS/acetate pH 7.5 and then subjected to sonication. After centrifugation, crude extract (CE) was incubated with nickel affinity column at 4°C for one hour. Total proteins in both CE and affinity purification fractions; flow-through (FT), washing steps (W1 to W4) and elution (E1 to E4) fractions were separated on SDS-PAGE 7.5%. Based on standard molecular weights, the apparent molecular weights of GlgE were estimated at 76 kDa and 72 kDa for E. lausannensis and W. chondrophila, respectively. c The optima of pH and d temperature of GlgE of E. lausannensis were determined by measuring the amount of orthophosphate released after the transfer of maltosyl moieties of M1P onto the non-reducing end of glucan chains. The optimum pH determination was carried out at 30°C in sodium acetate ((circle), pH 3.7; 4.8; 5.2), sodium citrate ((square), pH 4; 5; 6) and TRIS/HCl ((triangle) pH 6.8; 7.5; 7.7; 8; 8.8) buffers with a final concentration of 25mM. The optimum pH determination was carried out in the presence of 25 mM TRIS/HCl pH 6.8. e The uniprot accession numbers and amino acid sequences of recombinant proteins are displayed for each GlgE proteins. Amino acids underscored correspond to the His-tag and the att site produced during the cloning process in the expression vector.

Supplementary Figure 3: Proton-and phosphate-NMR analyses.
Reaction product purified following the incubation of Waddlia chondrophila GlgE with glycogen and orthophosphate. Complete 1D-1 H-NMR spectrum of maltoside-1-phosphate. α-anomer configuration of both glucosyl residues were characterized by their typical homonuclear vicinal coupling constants ( 3 J H1A,H2A and 3 J H1B,H2B ) with values of 3.5 Hz and 3.8 Hz respectively. A supplementary coupling constant was observed for α-anomeric proton of residue A as shown the presence of the characteristic doublet of doublet at 5.47 ppm. This supplementary coupling constant is due to the heteronuclear vicinal correlation ( 3 J H1A,P ) between anomeric proton of residue A and phosphorus atom of a phosphate group, indicating that phosphate group was undoubtedly O-linked on the first carbon of the terminal reducing glucosyl unit A. The value of this 3 J H1A,P was measured to 7.1Hz.

Supplementary Figure 4: FACE analysis of activity GlgE of Estrella Lausannensis.
Recombinant GlgE (3.5 nmol Pi.min -1 ) was incubated 1 hour and 16 hours at 30°C with 5 mM of malto-oligosaccharides composed of 0 to 7 glucose moieties (degree of polymerization: DP) and 0 mM or 1.6 mM of maltose-1-phosphate (M1P). After incubation, enzymatic reactions are stopped 5 min at 95°C. Malto-oligosaccharides are labeled with APTS and then separated according to their DP using capillary electrophoresis. Fluorescence is monitored as relative fluorescence units (RFU). As control, heat denatured GlgE activity was incubated 16 hours at 30°C with M1P and maltooligosaccharides.

Supplementary Figure 5: FACE analysis of activity GlgE of Waddlia chondrophila.
Recombinant GlgE (1.38 nmol Pi.min -1 ) was incubated 1 hour and 16 hours at 30°C with 5 mM of malto-oligosaccharides composed of 0 to 7 glucose moieties (degree of polymerization: DP) and 0 mM or 1.6 mM of maltose-1-phosphate (M1P). After incubation, enzymatic reactions are stopped 5 min at 95°C. Malto-oligosaccharides are labeled with APTS and then separated according to their DP using capillary electrophoresis. Fluorescence is monitored as relative fluorescence units (RFU). As control, heat denatured GlgE activity was incubated 16 hours at 30°C with M1P and maltooligosaccharides. Figure 6: a Native-PAGE containing glycogen reveals α-1,4 Glucanotransferase, elongation and hydrolytic activities of E. lausannensis GlgE. E. coli crude extract (CE) expressing GlgE-EL and purified GlgE-EL fraction (E1) were loaded onto native-PAGE containing 0.3% (w/v) of glycogen from bovine liver. Gel runs in ice pocket during 1h30 at 15 mA constant in TRIS/glycine buffer pH 8.8. After electrophoresis, native-gel was cut in three pieces and incubated overnight at room temperature with 10 mL 25 mM TRIS/acetate buffer pH 7.5 (Ø), 10 mL 25 mM TRIS/acetate buffer pH 7.5 and 1 mM of maltose-1-phosphate (M1P), 10 mL 25 mM TRIS/acetate buffer pH7.5 and 20 mM orthophosphate (Pi). Soaking native gel in iodine solution evidences GlgE activity. α-1,4 Glucanotransferase activity is visualized as brownish activity bands due to maltosyl reaction transfers catalyzed by GlgE-EL on the external glucan chains of glycogen particles. In presence of 1 mM M1P, the elongation activity is favored and consists in the maltosyl moieties transfer reactions of M1P onto non-reducing ends of external glucan chains of glycogen. The increase of long glucan chains leads to a strong iodine-glucan interaction observed as a dark activity band. At contrary, the hydrolytic reaction is conducted in the presence of 20 mM of Pi. GlgE-EL releases M1P from the non-reducing ends of external glucan chains of glycogen and α-1,6 linkages or branching points prevent the complete hydrolysis of glycogen particles.

Supplementary
Nevertheless, the resulting branched glucans escape from polyacrylamide gel leading to clear activity band in orange background. b Purification of branching enzyme (GlgB) activity of Waddlia chondrophila. The plasmid expression pET15b-GlgB-WC was transferred in ΔglgB Rosetta TM E. coli strain impaired in endogenous branching enzyme. After induction, crude extract (CE) was incubated for one hour with his-agarose beads at 4°C. Unbound proteins were eluted with 50 mM sodium acetate, 300 mM NaCl and 60 mM imidazole pH 7. After four washing steps, His-GlgB were eluted with 50 mM sodium acetate, 300 mM NaCl and 250 mM imidazole pH 7. Proteins in the flow through and elution fractions were separated on native-PAGE (7.5%) at 4°C (120 V, 15 mA). After electrophoresis, proteins were electrotransfered against a native-PAGE containing 0.3% (w/v) of potato starch using Trans-Blot® Turbo™ transfer system (Bio-Rad). Native-PAGE was then incubated overnight in 25 mM TRIS/acetate buffer pH 7.5 at room temperature. Branching enzyme activity is revealed as pink bands in blue background after soaking the gel in iodine solution (KI/I 2 ). c The uniprot accession number and amino acid sequence of recombinant proteins are displayed for GlgB protein. Amino acids underscored correspond to the His-tag and the att site produced during the cloning process in the expression vector.