Diapositive 1

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-? stimulate
hGBPs induction?
HeLa 229 cells
24h
Expression levels of hGBP1 and 2
qRT-PCR
Different doses of IFN-?
3
Is this induction sufficient to inhibit the
growth of Chlamydia trachomatis?
HeLa 229 cells
Infection
No IFN-?, 0.05ng/ml INF- ?, 0.5ng/ml INF- ?
Chlamydia trachomatis (strains L2, B e D)
24h
Immunofluorescence IFU assay
HeLa cells can induce hGBP1 and 2 in response to
higher doses of IFN-? 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
24h
Tranfection
Infection
C. trachomatis (serovar B)

• myc-GBP1
• D184N
• Helical domain

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
24h

• Immunostaining and confocal microscopy
• Morphometric analysis

24h
C. trachomatis (serovar L2, B ou D) at 1 MOI

• myc-hGBP1
• HÁ-hGBP2

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
24h
24h
C. trachomatis (B at 1 MOI

• myc-hGBP1
• HÁ-hGBP2

• 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

• GBP1 (wt)
• dominant negative mutant (D184N)
• helical domain only (HD)

Tranfection
24h
C. trachomatis (serovar B )
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-?
function?
HeLa 229 cells
Tranfection
siRNA for hGBP1 or siRNA control
Treatment with IFN-? (5ng/ml)
C. trachomatis (serovar B)
24h
24h
IFN-? and siRNA GBP1
IFN-? and siRNA control
- IFN-?
Better growth was obtained when the maximal
induction by IFN-? of hGBP1 was prevented by
siRNA.
9
Does the overexpression of hGBP1 potentiate IFN-?
function?
HeLa 229 cells
0.05ng/ml IFN-? C. trachomatis (serovar L2)

• myc-hGBP1
• Myc-hGBP1 D148N

24h
Immunofluorescence microscopy
18h
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
Infection
Different strains and species
24h
Morphometric analysis
myc-hGBP1
L2 lacks the cytotoxin gene B and D encode a
partial cytotoxin GPIC and MoPn posses a
full-length cytotoxin gene
C. muridarum
C. trachomatis
C. caviae
The intact cytotoxin may counteract the
anti-chlamydial effects of hGBP overexpression.
11
Conclusion
IFU
Mechanisms?
hGBP1 and 2
hGBP mark the inclusions for interaction with
degradative compartments autophagy machinery.
The hGBPs act as potentiators of IFN-? inhibition
of C. trachomatis growth, and may be the targets
of the chlamydial cytotoxin.