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, B -Endocervical epithelial cells were pre-treated or not with 40 µM CP4d for 2 h before 1182 addition of the indicated concentration of ionomycin (or an equivalent volume of DMSO) and 1183 0.5 µM BP. Six hours later whole cell lysates were analyzed by western blot. The membrane 1184 was first blotted with HRP-conjugated streptavidin to detect TG2 activity, then extensively 1185 washed and probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated 1186 secondary antibodies

, C -The same experimental procedure as described in (B) was applied to HeLa cells treated for 1188 48 h prior to ionomycin treatment (8 µM) with siRNA control or directed against TG2. 1189 1190 Figure 6. Optimal bacterial growth requires GFPT and prevents UDP-GlcNAc accumulation

, A -HeLa cells were infected or not (NI) with C. trachomatis (MOI = 1), then lysed 24 or 48 hpi

, After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-1193 O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies

, B -HeLa cells were infected or not with C. trachomatis (MOI = 1). Twenty-four hours later, 8 1197 µM ionomycin (or DMSO alone) and 0.5 mM BP were added. After 6 h of treatment

C. , After 1200 separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-GFPT 1201 and anti-actin antibody before revelation with HRP-conjugated secondary antibodies

, D -HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2. Two days 1203 later the cells were infected for the indicated times (MOI=0.3) before fixation, rupture of the 1204 cells and measurement of bacterial diameter. The mean diameter ± SD and p-values of 1205 Student

, Thirty hours later cells were fixed and analyzed by flow 1208 cytometry. The percentage of infected cells (top) and the mean fluorescence of the infected 1209 population (middle) ) ± SD are shown for at least four independent experiments. Duplicate 1210 wells were lysed and used to re-infect fresh HeLa cells to determine the bacterial titer 1211 (bottom). P-values of, E -HeLa cells treated for 48 h with siRNA targeting GFPT1 or not (siCTRL) were infected with 1207 C. trachomatis (MOI = 0.15)

, The increase in TG2 1213 expression and activity in cells infected with C. trachomatis results in the up-regulation of the 1214 expression of glucose transporters. Increasing quantities of glucose are thus imported in the 1215 host cytoplasm and redirected to the vacuole, where they fuel bacterial growth. Parallel to 1216 this transcriptional outcome, the transamidating activity of TG2 targets the host enzyme GFPT, 1217 thereby boosting the hexosamine biosynthesis pathway. The bacteria consume the resulting 1218 UDP-GlcNAc, or an intermediate along this pathway, TG2 activation in infection, p.1222, 1221.

, Figure EV1. TG2 is beneficial for bacterial development

, A -HeLa cells were pre-treated with the indicated concentrations of Cysteamine for 2 h before 1225 being infected with L2 incD GFP at MOI=0.15. Thirty hours later the cells were disrupted and 1226 bacterial titers (IFU) were determined by re-infecting fresh HeLa cells as described in the 1227 methods. The mean ± SD of three independent experiments are shown. P-values of Student

B. and T. , MEFs were infected with L2 incD GFP at MOI=0.15. Thirty h later the cells 1230 were disrupted and bacterial titers were determined by re-infecting fresh TG2 +/+ cells as 1231 described in the methods. The mean ± SD of five independent experiments and p-values from 1232 Student

, C -To measure bacterial adhesion TG2 +/+ and TG2 -/-MEFs were incubated at 4 °C for 4 h with 1234 L2 incD GFP at MOI=10 before being washed and fixed as described in the methods. The mean ± 1235 SD of three independent experiments are shown

D. and T. , MEFs were infected with L2 incD GFP at MOI=10 and fixed at the indicated 1237 time. Extracellular bacteria were differentially labeled as described in the methods, p.1238

, ± SD of three independent experiments, and p-values from Student's paired t-test, are shown, p.1239

E. Figure, Infection by C. muridarum activates TG2, which favors bacterial growth

, A -Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C. 1243 muridarum (MOI=1) in the presence or not of BP. Cell lysates were run on SDS-PAGE, proteins 1244 were transferred to a membrane and BP incorporation was revealed with HRP

, Duplicate wells were incubated further for 1251 a total of 48 h before the cells were fixed, permeabilized with 0.3% Triton-X100 and stained 1252 with rabbit antibodies against C. muridarum GroEL followed with A488-coupled anti-rabbit 1253 secondary antibodies. Samples were analyzed by flow cytometry, the percentage of infected 1254 cells (middle) and the mean fluorescence of the infected population (bottom) ± SD are shown 1255 for three independent experiments, p-values of Student, B -HeLa cells were transfected with siRNA against TG2 for 48 h before being infected in 1247 duplicates with C. muridarum at MOI=0.15

, Lines 1260 show example of measured RB diameters. Scale bar = 600 nm. RB diameters were measured 1261 using ImageJ on > 300 bacteria in one experiment. Each dot represents one RB, the mean value 1262 ± SD and p-value of Student's paired t-test are indicated, vol.1263, p.1265