THE USE OF PCR -RFLP OF THE

PHOSPHOLIPASE B GENE FOR THE

DIFFERENTIATION BETWEEN VARIETIES

AND MOLECULAR TYPES WITHIN

CRYPTOCOCCUS NEOFORMANSGEORGES 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

Department of Medicine, University of Sydney CIDM, Westmead Hospital

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, B, C, D and A/D

Eight 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

var. neoformans

var. gattii

Distribution world -wide mainly Europe tropical and subtropical

Environmental source

pigeon excreta decaying wood pigeon excreta decaying wood

Risk group Immuno compromised

Immuno compromised

Immuno competent

Clinical manifestation

meningitis 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

Serotype A (VN I)

Serotype B (VG I)

Serotype C (VG IV)

Serotype D (VN IV)

1980 bp

500 1000 1500

Hind III Ava I

RESULTSDigestion with Ava I

PCR-RFLP of VNI-VNIV isolates

VNI VNII VNIII VNIV

PCR-RFLP of VGI-VGIV isolates

VGI VGII VGIII VGIV

A A AD D B/C B/C B/C B/C

RESULTSDigestion with Hind III

PCR-RFLP of VNI-VNIV isolates PCR-RFLP of VGI-VGIV isolates

VNI VNII VNIII VNIV VGI VGII VGIII VGIV

A A AD D B/C B/C B/C B/C

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

ACKNOWLEDGMENTSThe 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