New Insight in the Pathogenesis

of LGV
Wim Sturm
University of KwaZulu-Natal

Chlamydia trachomatis
Obligate intracellular human pathogen
Causes trachoma, urogenital disease and LGV
2 biovars
based on differences in disease presentation + in vitro
cell growth characteristics

18 serovars
based on immunological cross-reactivity of MOMP

Oculogenital (OG) biovar

urogenital disease and conjunctivitis (D through K, Da,
and Ia)
ocular trachoma (A, B, Ba, and C)

Lymphogranuloma venereum (LGV) biovar

LGV (L1, L2, L2a, and L3)

Prevalence of LGV in patients with genital ulcer

disease in Durban
18
16
14
12
10
%
8
6
4
2
0
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Year

Lymphogranuloma venereum
skin of
genitalia

regional
lymphnodes

genital
ulcer

lymphadenitis

primary stage
cell death

lymphnode
destruction

secondary stage

tertiary stage

cell death

Lymphogranuloma venereum
skin of
genitalia

keratinocytes

primary stage

regional
lymphnodes
endothelium
macrophages
dendritic cells
lymphocytes
secondary stage

tertiary stage

Chlamydia and host-cells

early phase

inhibition of apoptosis

late phase

apo-necrosis

Growth of C. trachomatis in cells

HeLa cells or McCoy cells
35 37oC
Cell growth inhibitor
DEAE dextran
cyclohexamide
radiation

not comparable with

in-vivo situation

Chlamydia-host cell interaction

keratinocytes

33oC

37oC

endothelium
macrophages
dendritic cells
lymphocytes
cervical cells (ME180)

no cell growth inhibitors

Chlamydia strains
3 LGV laboratory strains
serovars L1, L2 and L3

3 LGV recent clinical isolates

serovar L2

Controls:
1 OG strain (serovar E)
Chlamydia free McCoy cell culture
cell free tissue culture medium

Cell lines
Keratinocytes
HaCaT

Endothelium
HUVEC

Macrophage
M43 cells

Dendritic cells
HB2 cells

Cell lines
Keratinocytes
HaCaT

Endothelium
HUVEC

Macrophage
M43 cells

Dendritic cells
HB2 cells

HaCaT cells
37oC

33oC

LGV serovar L2
(reference strain)

OG serovar E

ME 180 cells

37oC

LGV serovar L2
(reference strain)

OG serovar E

LGV in HaCaT cells at 33oC

EB in cytoplasma
outside inclusion body
only with LGV at 33oC

ME180 37C

HaCaT 37C

HaCaT 33C

Mean area occupied by Chlamydia per starting organism

15

Median size of chlamydial inclusions in HaCaT

cells at 33C from 2 to 5 days post infection
Area of chlamydial inclusions in HaCaT cells at 33C
5000

Area (m2)

4000

E
L1
L2
L3
US151
US162
US197

3000

2000

1000

0
2

Day

TUNEL assay in ME180

(TdT-mediated dUTP-biotin nick end labeling)

OG serovar E

LGV serovar L2

TUNEL assay

Cytotoxicity in HaCaT cells

Percent cytotoxicity exerted on HaCaT cells at 37C by C. trachomatis
Strain

12.00

E
L1
L2
L3
US151
US162
US197

10.00
8.00
6.00

Med p.tox

37oC

4.00
2.00
0.00
-2.00
-4.00
-6.00
-8.00
-10.00
-12.00

Percent cytotoxicity exerted on HaCaT cells at 33C by C. trachomatis

1

Day

12.00

Strain
E
L1
L2
L3
US151
US162
US197

10.00
8.00

33oC

Med p.tox

6.00
4.00
2.00
0.00
-2.00
-4.00
-6.00
-8.00
-10.00
-12.00
1

Day

Cytotoxicity in ME180 cells

Percent cytotoxicity exerted on ME-180 cells at 37C by C. trachomatis
Strain

12.00

E
L1
L2
L3
US151
US162
US197

10.00
8.00

Med p.tox

6.00
4.00
2.00
0.00
-2.00
-4.00
-6.00
-8.00
-10.00
-12.00
1

Day

Conclusions
The LGV reference strain L2 behaves very different from recent
clinical isolates of serovar L2
conclusions can only be drawn from work
with fresh clinical isolates
All strains tested grow faster at 37oC as compared to 33oC
LGV strains induce stronger apoptosis in HaCaT cells as
compared to the OG control
All LGV (but not the OG) strains, kill HaCaT cells at 33oC
through necrosis
cytotoxin ?
LGV specific

In HaCaT at 33oC, EB bodies of LGV strains escape from the

inclusions

Cell lines
Keratinocytes
HaCaT

Endothelium
HUVEC

Macrophage
M43 cells

Dendritic cells
HB2 cells

Extravasation

Uninfected HUVEC

HUVEC infected with L2

Conclusions
All C. trachomatis strains tested

grow in HUVEC cells

induce production of IL8 and MIP-1 by HUVEC.
induce the expression of ICAM-1 by HUVEC cells
induce cell death in HUVEC through apoptosis
induce necrotic cell death in HUVEC

There are quantitative differences between

strains
Limitation: this study used reference strains only

Cell lines
Keratinocytes
HaCaT

Endothelium
HUVEC

Macrophage
M43 cells

Dendritic cells
HB2 cells

Infected macrophages (M43) and dendritic cells (HB2)

Serovar E

Serovar L2

M43 cells

HB2 cells

Conclusions
Dendritic cells take up more chlamydia than
macrophages
Both LGV serovar L2 and OG serovar E induce
pro-inflammatory cytokine production in both
macrophages and dendritic cells
Both L2 and OG serovar E kill macrophages
and dendritic cells through necrosis and
apoptosis
Limitation: only 2 reference strains used

Lymphogranuloma venereum
skin of
genitalia

keratinocytes

primary stage

regional
lymphnodes
endothelium
macrophages
dendritic cells
lymphocytes
secondary stage

tertiary stage

Two new observations

C. trachomatis biovar LGV produces a
keratinocyte specific toxin
characterisation of toxin in progress

EB of LGV strains escapes from the inclusion

bodies in keratinocytes when grown at 33oC
allows for early escape to the lymphnodes ?

Acknowledgments
Dr. Bronwyn Joubert
Ikanyeng Dolly Seipone
Yoshen Moodley
Adele Cheddie