OF TRICHOMONAS VAGINALIS A N D F E S O D 6 GENETIC DIVERSITY

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PHYLOGENETIC RELATIONSHIPS

B E T W E E N T H E SIX SUPEROXIDE DISMUTASE PROTEINS ( F E S O D )

OF TRICHOMONAS VAGINALIS A N D F E S O D 6 GENETIC DIVERSITY

H W A N G U . W * , S H I N K.S.**, R Y U J . S . * * , M I N D . Y . * * & A H N M . H . * *

Summary:

The parasitic protozoan Trichomonas vaginalis is known to contain several types of Fe-containing superoxide dismutase proteins (FeSOD). Using three different methods of phylogenetic analysis, maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) methods, we examined the phylogenetic relationships among the six FeSOD (FeSOD1-FeSOD6) based on their amino acid sequences. All the analyses consistently suggested that the six proteins formed a monophyletic group implying that they probably be originated from an ancestral protein form through repeated duplication events. Although MP tree was totally unresolved, the NJ and ML trees revealed that FeSOD6 placed the most basal position and thus emerged earlier than the other five gene types during the evolution of T. vaginalis.

Phylogenetic relationships among the five remaining proteins were (FeSOD2, FeSOD3), (FeSOD4, (FeSODl, FeSOD5)| although weakly supported in terms of bootstrapping values. In addition to this, we newly designed two PCR primer specifically amplifying full-length FeSOD6 gene and examined its genetic diversity among

1 2 T. vaginalis isolates from five countries and three continents.

They had the same nucleotide sequences except those of three Korean isolates which showed one to three different nucleotides

KEY WORDS : Trichomonas vaginalis, FeSOD, molecular phylogeny, FeSOD6 genetic diversity

Résumé : RELATIONS PHYLOGÉNÉTIQUES ENTRE LES SIX PROTÉINES SUPEROXIDE DISMUTASE ( F E S O D ) DE TRICHOMONAS VAGINALIS ET LA DIVERSITÉ GÉNÉTIQUE DE FESOD6

Trichomonas vaginalis est un protozoaire parasite qui possède plusieurs protéines superoxide dismutase riches en fer (FeSOD). Les relations phylogénétiques entre les six protéines (FeSOD1-FeSOD6) ont été examinées en utilisant les méthodes d'analyse

phylogénétique suivantes : parcimonie, distance et maximum de vraisemblance. Toutes les analyses ont montré que les six protéines formaient un groupe monophylétique issu vraisemblablement d'une protéine ancestrale par des événements de duplication. Bien que l'arbre représentatif de l'analyse de parcimonie montre une irrésolution entre les six protéines, les arbres de distance et de maximum de vraisemblance placent la protéine FeSOD6 en position ancestrale. Les relations phylogénétiques entre les cinq autres protéines ne sont pas toutes bien défendues en terme de valeurs de bootstrap (robustesse aux nœuds), elles se présentent sous la forme (FeSOD2, FeSOD3), (FeSOD4, (FeSODl, FeSOD5jj. Deux nouvelles amorces, ont été définies pour amplifier l'intégralité du gène FeSOD6, ce qui a permis d'étudier sa diversité génétique à partir de 12 isolats de T . vaginalis en provenance de cinq pays et de trois continents. Il s'est avéré que toutes les séquences étaient identiques, à l'exception de trois isolats coréens qui présentaient une à trois bases de différence.

MOTS CLES : Trichomonas vaginalis, FeSOD, phylogénie moléculaire, diversité génétique de FeSOD6.

INTRODUCTION

T

richomonas vaginalis, a c o m m o n c a u s e o f vagi- nitis a n d e x o c e r v i c i t i s , is a m i c r o a e r o p h i l i c p r o - t o z o a n parasite that p o s s e s s e s m a n y c h a r a c t e - ristics o f a n a e r o b i c o r g a n i s m s , including sensitivity t o o x y g e n . A c c o r d i n g to a r e c e n t report ( W o r l d Health O r g a n i z a t i o n , 1 9 9 5 ) , t h e a n n u a l i n c i d e n c e o f t r i c h o - m o n i a s i s e x c e e d s 1 7 0 million c a s e s w o r l d w i d e . A d e - q u a t e d e f e n s e against o x i d a t i v e stress usually r e q u i r e s t h e p r e s e n c e o f a n u m b e r o f p r o t e c t i v e m e c h a n i s m s .

* Department of Biology, Teachers College, Kyungpook National Uni- versity, Taegu 702-701, Korea & Department of Parasitology, Yonsei University College of Medicine, Seoul 120-752, Korea.

** Department of Parasitology, Hanyang University College of Medi- cine, Seoul 133-791, Korea.

Hwang U.W. and Shin K.S. equally contributed to this work.

Correspondence: Dr. Jae-Sook Ryu.

Tel.: 82-2-2290-0683 - Fax: 82-2-2281-6519.

E-mail: jsryu@hanyang.ac.kr

T. vaginalis c o n s u m e s o x y g e n at l o w l e v e l s a n d o x y g e n c o n s u m p t i o n s y s t e m s are p r e s e n t in t h e c y t o - s o m e a n d in t h e h y d r o g e n o s o m e s (Ellis et al., 1 9 9 4 ) . S u p e r o x i d e d i s m u t a s e ( S O D ) c o n v e r t s s u p e r o x i d e a n i o n s ( 0^{2 -}) to m o l e c u l a r o x y g e n ( 0²) a n d h y d r o g e n p e r o x i d e ( H²0²) w h i c h , in turn, are m e t a b o l i z e d b y c a t a l a s e a n d p e r o x i d a s e ( B a n n i s t e r et al., 1 9 8 7 ) . T h r e e different S O D c l a s s e s , n a m e l y t h o s e o f F e S O D ( p r o - k a r y o t e s , plants, a n d p r o t o z o a ) , M n S O D ( p r o k a r y o t e s , m i t o c h o n d r i a , a n d c h l o r o p l a s t s ) , a n d C u / Z n S O D ( h i g h e r animals, a n d p l a n t s ) h a v e b e e n p u b l i s h e d to date. T h e y differ in their metal c o n t e n t s a n d in terms o f their sensitivities to c y a n i d e , azide, a n d h y d r o g e n p e r o x i d e ( M i c h e l s o n et al., 1 9 7 7 ; T a n n i c h et al., 1 9 9 1 ) . It is k n o w n that T. vaginalis has at least 7 F e S O D g e n e types, a m o n g w h i c h five g e n e t y p e s (from F e S O D l to F e S O D 5 ) h a v e b e e n partially c h a r a c t e r i z e d , but o n l y t h e F e S O D 6 g e n e t y p e has b e e n c o m p l e t e l y c h a r a c t e - rized ( V i s c o g l i o s i et al, 1 9 9 6 , 1 9 9 8 ) . Viscogliosi et al.

( 1 9 9 6 ) e x a m i n e d the p h y l o g e n e t i c relationships a m o n g

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Mémoire 37

Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/200209137

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HWANG U.W., SHIN K.S., RYU J.S., MIN D.Y. & AHN M.H.

F e S O D l - F e S O D 5 types b y the n e i g h b o r joining ( N J ) m e t h o d o n the basis o f partial a m i n o acid s e q u e n c e s , but did not study the phylogenetic position o f F e S O D 6 . In addition, n o o n e has r e s e a r c h e d the g e n e t i c diver

sity o f the F e S O D types a m o n g T. vaginalis isolates in parallel with geographical distribution studies.

In this study, w e reconstructed the p h y l o g e n e t i c rela

tionships o f the six F e S O D types to elucidate the phy

logenetic position o f the F e S O D 6 , and e x a m i n e d the d e g r e e o f g e n e t i c diversity o f the F e S O D 6 type in the 12 T. vaginalis isolates collected from five countries o n three different continents. Newly designed F e S O D 6 gene-specific PCR primers w e r e used to specifically amplify full-length F e S O D 6 g e n e .

MATERIALS AND METHODS

SAMPLE C O L L E C T I O N

A N D C E L L U L A R DNA E X T R A C T I O N

T

w e l v e T. vaginalis isolates w e r e o b t a i n e d from five geographically distinct regions (six Korean, o n e J a p a n e s e , o n e Chinese, o n e Australian, and three A m e r i c a n ) ( T a b l e I ) . T. vaginalis isolates w e r e grown axenically at 37° C in T Y M (Trypticase-Yeast extract-Maltose) m e d i u m and total g e n o m i c DNA w a s extracted from T. vaginalis isolates using the p h e n o l - chloroform extraction m e t h o d (Brindley et al., 1 9 9 3 ) .

P H Y L O G E N E T I C A N A L Y S I S

T o e x a m i n e phylogentic relationship a m o n g the six F e S O D t y p e s o f T. vaginalis, partial a m i n o a c i d s e q u e n c e s from the six F e S O D types o f T. vaginalis ( a c c e s s i o n no. Z 7 0 6 7 0 - Z 7 0 6 7 4 and A F 0 2 2 4 2 3 ) , T. gal- linarium ( Z 7 0 6 7 6 ) , Monocercomonas sp. ( C A A 9 4 5 2 4 ) , Hypotrichomonas acosta ( C A A 9 4 5 2 2 ) and Tritricho-

monas foetus ( A A C 4 7 7 3 4 ) w e r e retrieved from EMBL data b a n k . E x c e p t for the six from T. vaginalis, the remaining o n e s w e r e used as reference groups for phy-

Strains Geographic origin

NYH286 ATCC 50148

CDC85 America (Ohio)

RU393 ATCC 50142

IR78 Austria (Vienna)

YA1 Japan (Chiba)

KT19 China (Yonbyun)

KT4 Korea (Seoul)

KT11 Korea (Kuri)

KT12 Korea (Kuri)

KT18 Korea (Wonju)

KT38 Korea (Song-Nam)

KT40 Korea (Song-Nam)

Table I. - Trichomonas vaginalis isolates used in this study.

l o g e n e t i c analysis, o f w h i c h T. foetus was e m p l o y e d as an outgroup. Amino acid s e q u e n c e alignment o f F e S O D from T. vaginalis and other five trichomonads (Fig. 1) and nucleotide s e q u e n c e alignment o f F e S O D 6 g e n e s from twelve T. vaginalis isolates (Fig. 3 ) w e r e constructed b y the Clustal X multiple alignment pro

gram ( T h o m p s o n et al, 1 9 9 7 ) for phylogenetic analysis and s e q u e n c e c o m p a r i s o n . P h y l o g e n e t i c analysis w a s c o n d u c t e d u s i n g m a x i m u m p a r s i m o n y ( M P ) a n d n e i g h b o r joining (NJ) m e t h o d s o f PAUP* 4 . 0 beta 8 ( S w o f f o r d , 2 0 0 0 ) a n d m a x i m u m l i k e l i h o o d ( M L ) m e t h o d o f Puzzle 4 . 0 (Strimmer & von Haeseler, 1 9 9 6 ) . In MP and NJ analyses, bootstrapping analysis with 1,000 replicates w a s performed to estimate the d e g r e e o f c o n f i d e n c e o f e a c h clade. In ML, quartet puzzling ( 1 , 0 0 0 steps) was c o n d u c t e d for the s a m e purpose.

FULL-LENGTH PCR AMPLIFICATION

OF FESOD6 GENE AND DNA SEQUENCING

T h e entire F e S O D 6 g e n e was amplified b y PCR using Tacj DNA polymerase ( P r o m e g a , Madison, W i s c o n s i n ) . W e u s e d t w o newly d e s i g n e d primers, SODFL-1 ( 5 ' - ATG T T C ACA ATG G A G CAT CCT GCC-3', 5 - e n d position 1-24) and SODFL-2 (5'-TTA CAA ACC AGC AGC CTT-3', 3'-end position 5 6 8 - 5 8 5 ) , to specifically amplify t h e c o m p l e t e S O D c o d i n g r e g i o n o f t h e F e S O D 6 g e n e type from T. vaginalis isolates, by refer

ring to the report o f Viscogliosi et al. ( 1 9 9 8 ) . PCR w a s carried out using the following conditions: 9 4 ° C for five min ( o n e c y c l e ) ; and 3 0 c y c l e s o f 9 4 ° C for o n e min, 6 0 ° C for o n e min, and 7 2 ° C for o n e min; and finally 7 2 for 10 min ( o n e c y c l e ) . PCR products w e r e purified b y extraction from agarose gel using QIAXII ( Q i a g e n , Chatsworth, California). Purified PCR pro

ducts w e r e c l o n e d into the p G E M T-easy vector system following the manufacturer instructions ( P r o m e g a ) . T h e ligation mixture w a s u s e d to transform CaCl,- c o m p e t e n t E. coli T O P 10. White colonies w e r e selected b y the X - g a l / I P T G m e t h o d and plasmid DNA w a s iso

lated using the previously published alkaline m e t h o d ( S a m b r o o k et al, 1 9 8 9 ) . DNA s e q u e n c i n g was per

formed with an Li-COR a u t o m a t i c DNA s e q u e n c e r ( m o d e l 4 2 0 0 ) using T 7 forward and S P 6 reverse pri

mers.

RESULTS AND DISCUSSION

MONOPHYLETIC ORIGIN OF T. VAGINALIS FESOD AND PHYLOGENETIC POSITION OF FESOD6

T

he a m i n o acid s e q u e n c e s o f six F e S O D protein types o f T. vaginalis and F e S O D proteins from five trichomonad reference taxa w e r e aligned as s h o w n in Figure 1, w h i c h w a s used for the present p h y l o g e n e t i c analyses with MP, NJ, and ML m e t h o d s

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Fig. 1. - Amino acid sequence alignment of partial FeSODl - 6 of Trichomonas vaginalis and partial FeSOD from five reference taxa. This alignment was used for the phylogenetic analyses. T. vaginalis 1 - 6 , partial FeSODl - 6 of Trichomonas vaginalis, T gallinarum, FeSOD of Tetratrichomonas gallinarum; T. foetus, FeSOD of Trilrichomonas foetus, H acosta, FeSOD of Hypotrichomona acosta; M. sp., FeSOD of Monocercomonas sp. Dots indicate amino acids identical with those of the first line (FeSODl of T. vaginalis).

(Fig. 2 ) . As a result, T. vaginalis F e S O D proteins consis

tently formed a m o n o p h y l e t i c g r o u p with 7 4 %, 9 8 %, and 100 % bootstrap values in the MP, NJ, and ML ana

lyses, r e s p e c t i v e l y (Fig. 2 ) . Although relationships a m o n g six F e S O D types w e r e unresolved in the MP tree (Fig. 2A) e x c e p t a sister relation o f F e S O D 2 and F e S O D 3 (91 % bootstrapping value), the other two ana

lyses, NJ a n d ML (Figs. 2 B and 2 C ) , s h o w e d that F e S O D ô is placed at the most basal position. It implied that F e S O D 6 split off earlier than the others during the evolution o f T. vaginalis. In the NJ and ML analyses, p h y l o g e n e t i c relationships a m o n g six F e S O D types w e r e (FeSODô, ( F e S O D 4 , ( F e S O D l , F e S O D 5 ) , (FeSOD2, F e S O D 3 ) ) ) , but all the n o d e s w e r e not supported b y high bootstrapping values (Fig. 2 ) and the MP tree was unresolved. Notwithstanding these w e e k results, the relationships among F e S O D l ~5 types w e r e the same as t h o s e o b t a i n e d by Viscogliosi et al. ( 1 9 9 6 ) (using NJ m e t h o d and partial F e S O D l - F e S O D 5 s e q u e n c e s ) ,

but different in that the closest F e S O D type o f F e S O D 5 w a s found to b e F e S O D l and not F e S O D 4 but the bootstrapping value w a s relatively low in NJ ( 5 0 % ) and ML analyses ( 5 2 % ) .

SPECIFICITY O F S O D F L - 1 A N D - 2 P R I M E R S F O R A M P L I F I C A T I O N O F F E S O D6

A N D L O W G E N E T I C D I V E R S I T Y O F T H E F E S O D6 G E N E T h e c o m p l e t e nucleotide s e q u e n c e s o f F e S O D 6 from

12 7. vaginalis isolates collected from five geographi

cally distinct regions (six Korean, o n e J a p a n e s e , o n e Chinese, o n e Australian, and three American isolates) w e r e amplified by PCR using SODFL-1 and -2 primers ( s e e Materials and M e t h o d s ) w h i c h are specific to F e S O D 6 g e n e type. T h e o b t a i n e d PCR products w e r e c l o n e d and s e q u e n c e d . All the twelve F e S O D 6 g e n e s have the same lengths ( 5 8 5 b p ) without any indels. T h e nucleotide s e q u e n c e alignment o f the F e S O D 6 g e n e s

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T. vaginalis 1 QAYIDTANKLIVGSGFEGKSIEEVIQKAQGPLFNNVAQHFNHSFFWKCLSADKVAVPAKV [60]

T. vaginalis 2 L TPE..D..S.. [60]

T. vaginalis 3 L TPE..D..S.. [60]

T. vaginalis 4 L E [60]

T. vaginalis 5 L E [60]

T. vaginalis 6 .S P...I S...E H. [60]

T. gallinarum L. . . TM. . I. . A. S Y. .Q. . .R. .TPK. .El. . . . [60]

H. acosta R..V.M....VPE.PLN I..NST..I Y. .A. . .NS.T.Q.QEI.PS. [60]

M. sp. . . .V.MT. . .VP.TE. . . .T. .DIVKTSS..I ... I... Y D. .T.Q.QDI. .P. [60]

T. foetus . . . V. F . . . NVP . TE ....P...I....T A. . .N. .T.K.QE. . .G. [60]

T. vaginalis 1 ADALTKEFGSVEKFEETFTAKASTVFGSGWCYLYKNKEGKLEIGQYSNAANPVKDGFKPV [120]

T. vaginalis 2 ..V.ASN.E K S [120]

T. vaginalis 3 ..V.ASN.E K D. .C H..L [120]

T. vaginalis 4 Q D. .C H..L [120]

T. vaginalis 5 Q S H... [120]

T. vaginalis 6 .EL.K.N Q A. . . .T.D LT.I [120]

T. gallinarum KAV.E K.A.QG....L A. ... S F...L GV.I [120]

H. acosta . AF .I.H.K. . DD. KKD. V AA.QD.SIS.N HGF . I [120]

M. sp. QEF. . .H.Q. .D. .KTD.V AQ.ADKSIS.N NGF . I [120]

T. foetus .SF.A.H.E. .DN.KAQ.VQ AQ..DKTIS.N L GV.L [120]

T. vaginalis 1 LTVDT [125]

T. vaginalis 2 [125]

T. gallinarum .A... [125]

H. acosta [125]

M. sp. [125]

T. foetus .C... [125]

EVOLUTION OF TRICHOMONAS VAGINALIS F E S O D PROTEINS

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Fig. 2. - Phylogenetic relationships among six FeSOD types of Trichomonas vaginalis. (A) maximum parsimonious tree:

tree length = 150 steps, CI = 0.7800, RI = 0.6413, and RC = 0.5002. Of 125 total characters (inputted alignment shown in Figure 1), 56 are constant and 46 are parsimony-informative characters. ( B ) neighbor joining tree: distance matrix measured by mean character difference was used for the NJ analysis.

(C) maximum likelihood tree (Quartet puzzling tree): Dayhoff (Dayhoff etal., 1978) model was used as model of amino acid sequence substitution. Uniform rate of heterogeneity was assumed. Quartet puzzling was used to choose from the pos

sible tree topologies and to simultaneously infer support values for internal branches. The numbers above or below branches indicate bootstrapping values (1,000 replications) in (A) and ( B ) and quartet puzzling values (1,000 puzzling steps). Scale bars correspond to (A) 10 steps, ( B ) 0.05 sub

stitutions per amino acid, and (C) 0.1 substitutions per amino acid. Refer to the Figure 1 legend for the abbreviations.

HWANG U.W., SHIN K.S., RYU J.C., MIN D.Y & AHN M.H.

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EVOLUTION OF TRICHOMONAS VAGINALE F E S O D PROTEINS

TvSOD6 * ATGTTCACAATGGAGCATCCTGCCTACTTGAAGACTGGTCTTCCAGGCTTCCTCACACAG [60]

KT4 [60]

KT12 G... [60]

KT18 [60]

T V S 0 D 6 * CACGCTGTCGAGGTCCATGTTACAAAGCACCATCAGTCCTACATTGATACAGCTAACAAG [120]

KT4 [120]

KT12 ....TC [120]

KT18 [120]

T V S 0 D 6 * CTTATCGTTGGCTCTGGCTTCGAAGGCAAGCCAATTGAAGAAATCATCCAAAAGGCTCAG [180]

KT4 [180]

KT12 [180]

KT18 [180]

T V S O D 6 * GGCCCACTCTTCAACAACGTTGCCCAGCACTTCAACCACTCCTTCTTCTGGAAGTCCCTC [240]

KT4 [240]

KT12 [240]

KT18 [240]

T V S O D 6 * TCCGCTGAGAAGGTTGCTGTTCCAGCTCATGTTGCTGAGCTCCTCAAGAAGAACTTCGGC [300]

KT4 [300]

KT12 [300]

KT18 [300]

TVS0D6 * TCTGTCGAGAAGTTCCAGGAAACATTCACAGCTAAGGCTTCAACAGTCTTCGGCTCTGGC [360]

KT4 [360]

KT12 [360]

KT18 T [360]

TvSOD6 * TGGGCTTACCTTTACAAGACAAAGGACGGCAAGCTTGAGATCGGCCAGTACTCCAACGCT [420]

KT4 T [420]

KT12 [420]

KT18 G [420]

TVSOD6 * GCTAACCCAGTCAAGGATGGCCTTACACCAATTCTCACAGTCGATACATGGGAACATGCT [480]

KT4 [480]

KT12 [480]

KT18 [480]

TVSOD6 * TGGTACATCGACTACGAGAACAGAAAGGCTGAGTACTTCAAGAACTACTGGAACCACGTC [540]

KT4 [540]

KT12 [540]

KT18 [540]

TvSOD6* AACTGGAACTTTGTTGAGCAGAACTTAAAGGCTGCTGGTTTGTAAA [586]

KT4 [586]

KT12 [586]

KT18 [586]

Fig. 3- - Nucleotide sequence alignment of complete FeSOD6 genes from 12 Trichomonas vaginalis isolates collected from five geogra

phically distinct regions (six Korean, one Japanese, one Chinese, one Australian, and three American isolates) with previously reported one (TvSOD6). ' indicates that FeSOD6 gene sequences of the nine isolates (NYH286, CDC85, RU393, IR78, YA1, KT19, KT11, KT38, and KT40) are identical with that of the previously reported TvSOD6 gene (AF022423). Dots indicate nucleotide identical with those of the first line (TvSOD6). Refer to Table I for additional information of isolates.

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HWANG U.W., SHIN K.S., RYU J.S., MIN D.Y. & AHN M.H.

from t h e twelve isolates with a previously reported F e S O D 6 g e n e ( a c c e s s i o n n o . A F 0 2 2 4 2 3 ) is s h o w n in Figure 3. T h e o b t a i n e d s e q u e n c e c o m p a r i s o n s h o w e d that g e n e t i c differences o f T. vaginalis F e S O D 6 g e n e from t h e different geographical areas d o not exist with the e x c e p t i o n o f a small n u m b e r o f nucleotide c h a n g e s in t h e three K o r e a n isolates, K T 4 (1 b p ) , K T 1 2 ( 3 b p ) , and K T 1 8 ( 2 b p ) . This result also s h o w e d that t h e n e w primer set d e s i g n e d in t h e present study is highly s p e cific to t h e F e S O D 6 g e n e type.

CONCLUSION

P

h y l o g e n e t i c analysis w a s p e r f o r m e d with six F e S O D types revealed that all are m o n o p h y l e t i c and F e S O D 6 e m e r g e d earlier than t h e o t h e r types during t h e evolution o f T. vaginalis. It suggests that the six T. vaginalis F e S O D proteins could b e ori

g i n a t e d f r o m a n a n c e s t r a l p r o t e i n f o r m t h r o u g h r e p e a t e d duplication events. F e S O D 6 g e n e s e q u e n c e c o m p a r i s o n o f T. vaginalis isolates from various c o u n  tries a n d continents s h o w e d that the g e n e t i c diversity o f F e S O D 6 is very poor, since n o n e difference a p p e a red b e t w e e n t h e isolates from different countries a n d continents and curiously the very small differences ( o n e to three different n u c l e o t i d e s ) w e r e o b s e r v e d in three isolates from a same geographic origin. In addition, t w o newly d e s i g n e d PCR primers are highly specific to F e S O D 6 g e n e .

ACKNOWLEDGEMENTS

W

e heartily appreciate an anonymous reviewer for the critical a n d helpful c o m m e n t s . W e thank Dr. Kyung Y o o n (Dr. Y o o n ' s O b s t e trics & G y n e c o l o g y Clinic), Dr. M y e o n g - C h e o l Kim ( D e p a r t m e n t o f Obstetrics & G y n e c o l o g y , Y o n s e i Uni

versity Wonju College o f M e d i c i n e ) , Professor T c h u n - Y o n g Lee ( D e p a r t m e n t o f Urology, Hanyang Univer

sity C o l l e g e o f M e d i c i n e ) , Professor Akihiko Y a n o ( D e p a r t m e n t o f Parasitology Chiba University S c h o o l of Medicine) for the generous gift o f Trichomonas vagi

nalis. This work w a s supported b y the Hanyang Uni

versity, Korea, made in the program year o f 2000. UWH w a s s u p p o r t e d b y post-doc fellowship o f Brain K o r e a 21 Qan. 1, 2 0 0 0 - F e b . 2 8 , 2 0 0 1 ) .

BRINDLEY P.J., GAZZINELU R.T., DENKERS E.Y., DAVIS S.W., DIJBEY J.P., MARTINS M.C., SILVEIRA C , JAMIA L., WATERS A.P. & SHER A.

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