THE USE OF PCR -RFLP OF THE PHOSPHOLIPASE B GENE FOR THE DIFFERENTIATION BETWEEN VARIETIES AND MOLECULAR TYPES WITHIN CRYPTOCOCCUS NEOFORMANS GEORGES NICOLAS LATOUCHE 1,2, TANIA SORRELL 1,2 and WIELAND MEYER 1,2 1 Centre for Infectious Diseases and Microbiology, Molecular Mycology Laboratory, Westmead Hospital, Westmead, NSW 2145; 2 Department of Medicine, The University of Sydney, Sydney, NSW 2006 Department of Medicine, University of SydneyCIDM, Westmead Hospital

INTRODUCTION u C. neoformans is a human pathogenic yeast u The species can be divided into: u Three varieties u C. neoformans var. grubii u C. neoformans var. neoformans u C. neoformans var. gattii u Five serotypes u A, B, C, D and A/D u Eight molecular types based on PCR-Fingerprinting u VNI-VNIV and VGI-VGIV

Groupings in C. neoformans

Introduction (Cont)

INTRODUCTION (Cont) u The differentiation between yeast strains is of epidemiological and taxonomical importance u A molecular method differentiating between serotypes would expedite the processing of new isolates received in our laboratory u A method able to differentiate var. grubii from the other varieties would go a long way towards validating this new varietal status

INTRODUCTION (Cont) u 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 u 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) u The PLB gene of serotype A and serotype D have been submitted to GeneBank by others u In our laboratory we have isolated, cloned and sequenced the PLB gene of serotypes B and C u The PLB gene is one of few genes that has been sequenced in its entirety for all four serotypes

AIMS u To develop a PCR-RFLP method based on the PLB gene to differentiate between varieties, serotypes and molecular types of Cryptococcus neoformans u 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 Hind IIIAva I

RESULTS Digestion with Ava I PCR-RFLP of VNI-VNIV isolates VNIVNIIVNIIIVNIV PCR-RFLP of VGI-VGIV isolates VGIVGIIVGIIIVGIV A A AD D B/C B/C B/C B/C

RESULTS Digestion with Hind III PCR-RFLP of VNI-VNIV isolatesPCR-RFLP of VGI-VGIV isolates VNIVNIIVNIIIVNIV VGIVGIIVGIIIVGIV A A AD D B/C B/C B/C B/C

SUMMARY u Digestion with Ava I distinguished between molecular types and varieties u Digestion with Hind III allowed for differentiation of the varieties only u The differentiation between serotypes was not achieved with either enzyme u The RFLP profile of serotype A/D isolates was a combination of serotype A and serotype D profiles

CONCLUSIONS u The PCR-RFLP of the PLB gene allows for the distinction between varieties and molecular types u The distinction between serotypes B and C was not achieved due to their close genetic relatedness u The traditional PCR-RFLP on the ITS fragment results in one profile for all C. neoformans isolates u 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 u The Community Health and Tuberculosis Association for the Harry Windsor Postgraduate Scholarship to NL u The Millenium Foundation for the Top-Up grant to NL u The NH&MRC for Grant # to WM The authors would also like to thank Matthew Huynh for his assistance