1. Objective: To characterize the relationship between hGBPs and chlamydial growth in cultured cells. Thais Herrero Geraldino

2. Does treatment of HeLa cells with IFN-γ stimulate hGBPs induction? HeLa 229 cells Different doses of IFN-γ 24h Expression levels of hGBP1 and 2 qRT-PCR

3. Is this induction sufficient to inhibit the growth of Chlamydia trachomatis? HeLa 229 cells Infection Chlamydia trachomatis (strains L2, B e D) 24h No IFN-γ, 0.05ng/ml INF- γ, 0.5ng/ml INF- γ Immunofluorescence IFU assay HeLa cells can induce hGBP1 and 2 in response to higher doses of IFN-γ treatment which correlated well with more efficient inhibition of growth of different C. trachomatis strains.

4. Could hGBPs associate with membranous structures? HeLa 229 cells Tranfection myc-GBP1 D184N Helical domain Infection C. trachomatis (serovar B) 24h Immunofluorescence microscopy These results indicate that the helical domain is sufficient to localize hGBP1 to the membrane.

5. What is the potential anti-Chlamydial activity of hGBP1 and 2? HeLa 229 cells Tranfection myc-hGBP1 HÁ-hGBP2 24h C. trachomatis (serovar L2, B ou D) at 1 MOI 24h Immunostaining and confocal microscopy Morphometric analysis These data demonstrate that overexpression of hGBP1 is sufficient to produce a noticeable and statistically significant inhibition of chlamydial growth.

6. What is the potential anti-Chlamydial activity of hGBP1 and 2? HeLa 229 cells Tranfection myc-hGBP1 HÁ-hGBP2 24h C. trachomatis (B at 1 MOI 24h Morphometric analysis The overexpression of hGBP2, as well as hGBP1, may have an anti-chlamydial activity.

7. Is the GTPase domain required for hGBP function? HeLa 229 cells Tranfection GBP1 (wt) dominant negative mutant (D184N) helical domain only (HD) C. trachomatis (serovar B ) 24h Morphometric analysis The deletion mutant, and subtler D184N point mutant lost the ability to inhibit chlamydia growth.

8. Does the overexpression of hGBP1 potentiate IFN-γ function? HeLa 229 cells Tranfection siRNA for hGBP1 or siRNA control 24h Treatment with IFN-γ (5ng/ml) C. trachomatis (serovar B) 24h - IFN-γ + IFN-γ and siRNA control + IFN-γ and siRNA GBP1 Better growth was obtained when the maximal induction by IFN-γ of hGBP1 was prevented by siRNA.

9. Does the overexpression of hGBP1 potentiate IFN-γ function? HeLa 229 cells myc-hGBP1 Myc-hGBP1 D148N 18h 0.05ng/ml IFN-γ + C. trachomatis (serovar L2) 24h Immunofluorescence microscopy Myc-hGBP1 Myc-hGBP1 D148N The data indicate that the sub-inhibitory concentration of IFN-y could be sufficient in inhibiting chlamydial growth when accompanied by hGBP1 overexpression.

10. Are there a correlation between sensitivity GBP overexpression and the presence of citotoxin? HeLa 229 cells Tranfection myc-hGBP1 Infection Different strains and species 24h Morphometric analysis C. trachomatis C. caviae C. muridarum L2 lacks the cytotoxin gene; B and D encode a partial cytotoxin; GPIC and MoPn posses a full-length cytotoxin gene The intact cytotoxin may counteract the anti-chlamydial effects of hGBP overexpression.

11. Conclusion hGBP1 and 2 IFU hGBP mark the inclusions for interaction with degradative compartments – autophagy machinery. Mechanisms? The hGBPs act as potentiators of IFN-γ inhibition of C. trachomatis growth, and may be the targets of the chlamydial cytotoxin.