Transcript Molecular Characterisation of Cryptococcal Phospholipases

Department of Medicine, University of Sydney
CIDM, Westmead Hospital
THE USE OF PCR -RFLP OF THE
PHOSPHOLIPASE B GENE FOR THE
DIFFERENTIATION BETWEEN VARIETIES
AND MOLECULAR TYPES WITHIN
CRYPTOCOCCUS NEOFORMANS
GEORGES NICOLAS LATOUCHE1,2, TANIA SORRELL1,2 and
WIELAND MEYER1,2
1Centre
for Infectious Diseases and Microbiology, Molecular Mycology Laboratory, Westmead
Hospital, Westmead, NSW 2145;
2Department of Medicine, The University of Sydney, Sydney, NSW 2006
INTRODUCTION
 C.
neoformans is a human pathogenic yeast
 The
species can be divided into:
 Three
varieties
 C.
neoformans var. grubii
 C. neoformans var. neoformans
 C. neoformans var. gattii
 Five
serotypes
 A,
 Eight
B, C, D and A/D
molecular types based on PCR-Fingerprinting
 VNI-VNIV
and VGI-VGIV
Groupings in C. neoformans
SEROTYPE
VARIETY
MOLECULAR TYPE
A
grubii
VNI, VNII
B
gattii
VGI-VGIV
C
gattii
VGI-VGIV
D
neoformans
VNIV
AD
Hybrid
VNIII
Introduction (Cont)
var.
grubii
Distribution
Environmental
source
Risk group
Clinical
manifestation
world-wide
var.
neoformans
var.
gattii
mainly Europe
tropical and
subtropical
pigeon excreta
pigeon excreta
decaying wood
Immuno
compromised
meningitis
decaying wood
Immuno
compromised
Immuno
competent
meningitis
pulmonary
cryptococcomas
INTRODUCTION (Cont)
 The
differentiation between yeast strains is of
epidemiological and taxonomical importance
A
molecular method differentiating between serotypes
would expedite the processing of new isolates
received in our laboratory
A
method able to differentiate var. grubii from the
other varieties would go a long way towards
validating this new varietal status
INTRODUCTION (Cont)
 Phospholipase
B (PLB) breaks down phospholipids
and may be involved in the ability of the pathogen to
gain access to the blood stream from the lung
 The
PLB gene has recently been shown to play a role
in the virulence of C.neoformans var. grubii in mouse
model of Cryptococcosis
INTRODUCTION (Cont)
 The
PLB gene of serotype A and serotype D
have been submitted to GeneBank by others
 In
our laboratory we have isolated, cloned and
sequenced the PLB gene of serotypes B and C
 The
PLB gene is one of few genes that has been
sequenced in its entirety for all four serotypes
AIMS
 To
develop a PCR-RFLP method based on the
PLB gene to differentiate between varieties,
serotypes and molecular types of Cryptococcus
neoformans
 To
apply this method to isolates of known
variety, serotype and molecular type
METHODS
1980 bp
500
1000
1500
Serotype A (VN I)
Serotype B (VG I)
Serotype C (VG IV)
Serotype D (VN IV)
Hind III
Ava I
RESULTS
Digestion with Ava I
VNI
A
VNII
VNIII
VNIV
A
AD
D
PCR-RFLP of VNI-VNIV isolates
VGI
B/C
VGII
B/C
VGIII
B/C
VGIV
B/C
PCR-RFLP of VGI-VGIV isolates
RESULTS
Digestion with Hind III
VNI
VNII
A
A
VNIII
VNIV
VGI
VGII
VGIII
VGIV
AD
D
B/C
B/C
B/C
B/C
PCR-RFLP of VNI-VNIV isolates
PCR-RFLP of VGI-VGIV isolates
SUMMARY
 Digestion
with Ava I distinguished between molecular
types and varieties
 Digestion
with Hind III allowed for differentiation of
the varieties only
 The
differentiation between serotypes was not
achieved with either enzyme
 The
RFLP profile of serotype A/D isolates was a
combination of serotype A and serotype D profiles
CONCLUSIONS
 The
PCR-RFLP of the PLB gene allows for the
distinction between varieties and molecular types
 The
distinction between serotypes B and C was not
achieved due to their close genetic relatedness
 The
traditional PCR-RFLP on the ITS fragment results
in one profile for all C. neoformans isolates
 The
ploidy of serotype AD strains at the PLB and other
virulence loci needs to be further investigated
ACKNOWLEDGMENTS
The authors would like to acknowledge the following
organisations for their financial support
 The
Community Health and Tuberculosis Association
for the Harry Windsor Postgraduate Scholarship to NL
 The
Millenium Foundation for the Top-Up grant to NL
 The
NH&MRC for Grant # 9937187 to WM
The authors would also like to thank Matthew Huynh
for his assistance