Objective: To characterize the relationship between hGBPs and chlamydial growth in cultured cells.

Thais Herrero Geraldino

Does treatment of HeLa cells with IFN-γ stimulate hGBPs induction?

HeLa 229 cells

Different doses of IFN-γ24h Expression levels of hGBP1

and 2qRT-PCR

Is this induction sufficient to inhibit the growth of Chlamydia trachomatis?

HeLa 229 cells

Infection Chlamydia trachomatis (strains L2, B e D)

24hNo IFN-γ, 0.05ng/ml INF- γ, 0.5ng/ml INF- γ

ImmunofluorescenceIFU 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.

Could hGBPs associate with membranous structures?

HeLa 229 cells

Tranfection • myc-GBP1• D184N• Helical domain

Infection C. trachomatis (serovar B)

24hImmunofluorescence

microscopy

These results indicate that the helical domain is sufficient to localize hGBP1 to the membrane.

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.

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.

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.

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.

Does the overexpression of hGBP1 potentiate IFN-γ function?

HeLa 229 cells

• myc-hGBP1

• Myc-hGBP1 D148N

18h0.05ng/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.

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. muridarumL2 lacks the cytotoxin gene;

B and D encode a partial cytotoxin;

GPIC and MoPn posses a full-length cytotoxin geneThe intact cytotoxin may counteract the anti-chlamydial effects of hGBP overexpression.

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.