Post on 15-Jan-2016
description
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.