GENOME ORGANISATION AND GENETIC EXCHANGE ME CHANISMS IN NEISSERIA G O N O R R H O E A E : POSSIBLE IMPLICATIONS FOR CELL SURFACE LABILITY AND PA THOGENICITY

AKADEMI SK AVHANDL I NG

Aom me.d vzdeAb'ónllgt t ll lò tà n d av Re.ktonAämbe£el vid Umeå UnlveAAllel faön avläggande, av falloso fale. do klo nò examen kommen, a l t ofafaenltlgt faönj>vanaA l faonelaònlngòòalen, Inòtltwtlonen faön. Ihlknobiologi, fanedagen den 13 juvu. 19SO kl 09.00.

avLENA NORLANDER

ABSTRACTGENOME ORGANISATION AND GENETIC EXCHANGE MECHANISMS IN NEISSERIA GONORRHOEAE: POSSIBLE IMPLICATIONS FOR CELL SURFACE LABILITY AND PATHOGENICITY

Lena N o r l a n d e r , Depa r tmen t o f M i c r o b i o l o g y , U n i v e r s i t y o f Umeå, Sweden

N e i s s e r i a g o n o r r h o e a e shows a h igh d e g r e e o f l i a b i l i t y o f c e l l s u r f a c e compo nen t s , such a s p i l i and c e r t a i n o u t e r membrane p r o t e i n s , which r e s u l t s in t h e a p p e a r a n c e o f s e v e r a l c h a r a c t e r i s t i c c o l o n i a l t y p e s . The s e g r e g a t i o n be tween c o l o n i a l v a r i a n t s o c c u r s q u i t e f r e q u e n t l y and i s c o r r e l a t e d w i t h t h e c e s s a t i o n o f g rowth and t h e a u t o l y t i c a l r e l e a s e o f DNA. C o lo n i a l s e g r e g a t i o n i s s u g g e s t e d t o be a b a s i c g e n e t i c e v e n t , such as an i n v e r s i o n , wh ich i s a m p l i f i e d in a p o p u l a t i o n by an e f f i c i e n t t r a n s f o r m a t i o n sy s t em .

D i f f e r e n t s u r f a c e component s o f gon ococc i c o n f e r s p e c i f i c p r o p e r t i e s t o t he c e l l s . For example , t h e p r e s e n c e o f p i l i , a c h a r a c t e r i s t i c o f v i r u l e n t c o l o n i a l t y p e s , r e s u l t s in a more h y d ro p ho b i c and n e g a t i v e l y ch a r ge d c e l l s u r f a c e .The c o m p o s i t i o n o f t h i s s u r f a c e i s d e p en d en t on t h e g rowth m i l i e u . Growth a t pH 6 . 0 , f o r example , compared t o h i g h e r pH, i n c r e a s e s t h e n e g a t i v e s u r f a c e c h a r g e o f a l l c o l o n i a l v a r i a n t s .

G e n e t i c exchange mechanisms in go ncocc i we re s t u d i e d in d e t a i l . I t was found t h a t a l a r g e gono coc ca l p l a s m id m e d i a t e d t h e t r a n s f e r o f p l a s mid but no t chromosomal DNA. The p r e s e n c e o f t h i s p l a sm id c o n f e r r e d a p a r t i a l compe t ence f o r t r a n s f o r m a t i o n . These and o t h e r d a t a s u g g e s t a r o l e f o r p l a s m i d s in t h e go noc occ a l t r a n s f o r m a t i o n p r o c e s s . No rm a l ly , o n l y p i l i a t e d v a r i a n t s a r e t r a n s f o r m a b l e . The d i f f e r e n c e in t r a n s f o r m a t i o n e f f i c i e n c y between p i l i a t e d and n o n p i l i a t e d d e r i v a t i v e s i s due b o t h t o a d i f f e r e n c e in u p t a k e o f DNA and i t s s u b s e q u e n t p r o c e s s i n g .

P i l i a t e d go no coc ca l c e l l s t a k e up p l a s mi d f r a g m e n t s o f homologous a s we l l a s o f h e t e r o l o g o u s o r i g i n . These f r a g m e n t s , which have been t a k e n up i n t o a DNase r e s i s t a n t s t a t e , can be r e i s o l a t e d f rom ly se d c e l l s . A u t o r a d i o g r a p h y o f such l y s a t e s i n d i c a t e s a p r e f e r e n t i a l u p t a k e o f homologous f r a g m e n t s from a m i x t u r e o f h e t e r o - and homologous p l a s mi d d i g e s t s . A l s o , t h e l a r g e r f r a g m e n t s a r e t a k e n up more r e a d i l y t h an t h e s m a l l e r o n e s .

Gonococci a r e t h o u g h t t o have a DNA r e c o g n i t i o n sy s t em t h a t f a v o u r s homologous DNA. The n a t u r e o f t h i s sy s t em i s unknown. The d i s c r i m i n a t i o n a g a i n s t h e t e r o l o g o u s DNA a t t h e l e v e l o f DNA u p t a k e was n o t n e a r l y a s e f f i c i e n t a s t h a t found in Haemophi1 u s , p e rh a p s i n d i c a t i n g a d i f f e r e n t mechan i sm.

Gonococci we re found t o have m o d i f i e d c y t o s i n e r e s i d u e s in t h e i r r e c o g n i t i o n s e q u e n c e s f o r t h e r e s t r i c t i o n e n d o n u c l e a s e s H a e l l , H a e I I I , and Sac I I . These enzymes a r e i s o s c h i z o m e r s o f t h e gonoc occ a l e n d o n u c l e a s e s Ngol , N g o l l , and N g o I I I . The t h i r d enzyme was i d e n t i f i e d in t h i s work . Because o f t h e s e f i n d i n g s i t i s s u g g e s t e d t h a t gon ococc i p o s s e s s a h o s t r e s t r i c t i o n m o d i f i c a t i o n sy s t em . In a d d i t i o n , two o u t o f f i v e gon ococca l s t r a i n s had t h e a b i l i t y t o mod i fy t h e a d e n i n e o f t h e s e q ue n ce -GATC-. V a r i a t i o n s in m o d i f i c a t i o n p a t t e r n s may have i m p l i c a t i o n s f o r t h e p h y s i o l o g y and t he p a t h o g e n i c i t y o f g o n o c o c c i .

K e y w o r d s : N e i s s e r i a g o n o r r h o e a e , c o l o n i a l s e g r e g a t i o n , g e n e t i c exchange ,m o d i f i c a t i o n , r e s t r i c t i o n e n d o n u c l e a s e

ISSN 03^6-6612

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New S e r i e s No. 59From th e Depar tment o f M i c ro b io l o g y U n i v e r s i t y o f Umeå, Umeå, Sweden

GENOME ORGANISATION AND GENETIC EXCHANGE MECHANISMS IN NEISSERIA G O N O R R H O E A E : POSSIBLE IMPLICATIONS FOR CELL SURFACE LABILITY AND PATHOGENICITY

BY

Le n a No r l a n d e r

Umeå Un i v e r s i t e t

Umeå 1980

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New S e r i e s No. 59

E d i t o r : t h e Dean o f t h e F a c u l t y o f Med i c ine

ISSN 0346-6612

To th e Gonococchi

The gfieat txagedy ofi Science -

the. t>hcytng ofi a beauttfaut

kypotheA-ù) by an ugly ßact.

T. H. Haxtey

CONTENTS

PREFACE 6

I. INTRODUCTION

1. C o lo n ia l r e l a t i o n s h i p to v i r u l e n c e 7

2. The c e l l en ve lo p e o f Ne i s s e r i a gonorrhoeae 8

3. The s u r f a c e p r o t e i n s o f gonococcal c o l o n i a l v a r i a n t s 9

4 . G on o co c c i -h o s t c e l l i n t e r a c t i o n 11

5. D eo x y r i b o n u c l e i c ac id c o n t e n t 13

6. Trans format ion in gonococc i 15

I I . SUMMARY OF THE PRESENT WORK

1. Co lon ia l v a r i a t i o n in N e i s s e r i a gonorrhoeae 18

2. Phys i co -c hem ic a l nature o f the c e l l s u r f a c e 20

o f d i f f e r e n t gonococcal v a r i a n t s

3. The r o l e o f th e 24 .5 x 10^ d a l t on plasmid 23

4 . R e s t r i c t i o n m o d i f i c a t i o n in gonococc i 26

5. DNA uptake in N e i s s e r i a gonorrhoeae 28

I I I . GENERAL DISCUSSION AND CONCLUSIONS 30

ACKNOWLEDGMENTS 40

REFERENCES 41

6

PREFACE

This t h e s i s i s based on th e f o l l o w i n g p u b l i c a t i o n s and

manuscr ip t s :

I Norlander , L . , J . D a v ie s , A. N o r q v i s t , and S. Normark. 1979.

Genet i c b a s i s f o r c o l o n i a l v a r i a t i o n in N e i s s e r i a gon or rh oe a e .

J. B a c t e r i o l . T38: 762 -76 9 .

II Magnusson, K-E. , E. Kih l s t röm, L. Nor lander , A, N o r q v i s t ,

J . D a v ie s , and S. Normark. 1979, E f f e c t o f co lo n y type and

pH on s u r f a c e charge and h yd r op h o b ic i ty o f N e i s s e r i a go n or rh oe a e .

I n f e c t . Immun. 26: 397 -40 1 .

I I I Nor lander , L . , J. D a v ie s , and S. Normark. 1979. Gene t i c

exchange mechanisms in N e i s s e r i a g ono rrh oea e . J. B a c t e r i o l .

138 : 756 -761 .

IV Norlander , L . , J. K. D a v ie s , P. Hagblom, and S, Normark. 1980.

D eo x yr ib on u c l e i c a c id m o d i f i c a t i o n s and r e s t r i c t i o n en don uc l eas e

produc t ion in N e i s s e r i a go no rrh oe ae . J. B a c t e r i o l . ( sub mi t t ed

f o r p u b l i c a t i o n ) .

V Norlander , L . , and S. Normark. 1980. D eo x yr i bo nu c l e as e

r e s i s t a n t uptake o f plasmid DNA in N e i s s e r i a go n o rrh o ea e .

7

I. INTRODUCTION

1:1 Colonial relationship to virulence Neisseria gonorrhoeae is the etiological agent of gonorrhea, one of the most common infectious diseases of our time and moreover, a disease that has been known since antiquity.

The gonococci are gram negative diplococci which readily undergo autolysis (13, 19, 46). During subculturing

the gonococci give rise to characteristic colonial types. Four main, morphologically distinct, colonial variants - types 1, 2, 3 and 4 (Tl, T2, T3, T4) - were initially described by Kellogg et aj.. (37) . A number of intermediate colonial variants have later been identified (6, 32). It has been firmly established that there exists a- strong correlation between the colonial morphology in vitro and the degree of virulence a particular strain of Neisseria gonorrhoeae displays in vivo. The Tl and T2 colony variants which predominate in recent clinical isolates (37, 68) appear to be virulent both in human volunteers (36, 37) and in various animal models (4, 5, 52).

On non-selective subculturing in vitro these strains give rise to large numbers of T3 or T4 colonies, which are relatively avirulent. Furthermore, a considerable variation between the virulent colony types, Tl and T2, as well as between the avirulent T3 and T4 colonial variants occurs (6, 32, 37). In addition, a reversion from avirulent

colonial morphology to virulent form has been noticed both in vivo (36, 37, 80, 82) and in vitro (17, 38). Reversion, however, is an infrequent event in vitro and seems to be a strain-related phenomenon. This process seems to be enhanced in several of the animal model systems.

1:2 The cell envelope of Neisseria gonorrhoeae The cell envelope of gonococci consists of the cytoplasmic membrane, the peptidoglycan layer, and the outer membrane (49). The peptidoglycan structure is similar to that of E. coli except that gonococci lack covalently attached lipoproteins when grown at pH 7.2 (21, 87). The peptido­glycan when prepared from gonococci grown at a lower pH had, in addition, some other proteins associated with it (18). Gonococci readily undergo autolysis, which is associated with- peptidoglycan hydrolysis. An N-acetyl- muramyl-L-alanine amidase activity has been detected in gonococci, but other types of hydrolytic systems exist as well (20). The autolysis is suppressed at pH 6.0, where the rate of peptidoglycan hydrolysis is also markedly reduced compared to that at a higher pH (85).

Gonococci possess an outer membrane with a composition similar to that of other gram negative bacteria (34, 87) .It consists of lipopolysaccharides (LPS), phospholipids, and proteins. The LPS isolated from gonococci lacked the

9

0-antigen polysaccharide and were therefore of the R-type. No qualitative differences have been observed between Tl LPS and T4 LPS (86).

1:3 The surface proteins of gonococcal colonial variants Variations in the composition of the cell surface have been shown to be the basis for the different colonial variants. The difference between the virulent Tl and T2 variants and the avirulent T3 and T4 colonial types is that only the former possess pili (33, 78). A large part of the variation in colonial morphology can be correlated with specific changes in a series of outer membrane proteins, the colony opacity associated (COA) proteins (41, 42, 75, 76). It has recently been shown that pili isolated from opaque variants differed in their molecular weight from pili purified from transparent colonies of the same strain (60).

Three major outer membrane proteins have been reported (22, 23, 34, 35). Protein I is present in both the parent strains and in variants derived by repeated subculturing. Protein II, however, is reported to be present only in laboratory-derived strains (22). Protein III, whose presence has only been demonstrated in one gonococcal strain, is lost on repeated subculturing (23) .

Transparent colony types (Tl and T4) seem to contain only one major surface protein, protein I (41). The

10

highly coloured, opaque, T2 and T3, variants were shownto produce additional proteins (76, 83). Moreover, aseries of intermediate opacity colonies has been identified

&(75). A number of additional surface proteins, protein Ila , lib , lie has been correlated to these intermediate colonial opacity variants. However, the number of extra proteins has not been quantitatively related to colony opacity (41).

Table 1 summarizes the correlation between the main colonial variants and the presence of pili and protein composition of the outer membrane.

Table 1 Protein components of gonococcal colonialvariants (22, 23)

Colonyvariant

Pigmentation Pili Protein I 36,500 dal

Protein II . 24,000 dal

Protein III 60,000 dal

Tl - + + - + -Tly + + + + + -T2 + + + + + -T3 + - + + + -T4y + - + + + -T4 - - + - + -

a) Protein II showed an apparent increase in molecular weight on boiling in SDS (protein II ; 29,000) . Intermediate opacity variants contained additional surface proteins (Ila % 28,500; lib*, 28,000; lie* 27,500) (41).

11

1:4 Gonococci-host cell interactionThe interaction of gonococci with host-cell surfaces is a critical early stage in the pathogenesis of gonorrhoea. Binding of Neisseria gonorrhoeae to tissue culture cells, human sperm, human vaginal epithelial cells and buccal mucosal cells in in vitro systems appears to be influenced primarily by the presence or absence of pili on the bacteria (31, 47, 53, 72, 74). It has been demonstrated with purified radioactively labelled pili that they attach most efficiently to cells which are histologically the most similar to the actual sites of gonococcal infection (51).

The degree of attachment of gonococci has been found to increase upon lowering the pH to below 7.2. This has implications for the establishment of infection Jin vivo, since the pH at the site of infection is around pH 5.5. Furthermore, opaque colony variants, which lack pili but possess additional outer membrane proteins, have a considerable advantage in attachment to buccal epithelial cells compared to non-opaque types (42, 74).

It has also been shown that the interaction betweenhuman polymorphonuclear leucocytes and Neisseria gonorrhoeaeis primarily determined by a protein surface factor,the leucocyte-association factor (LA-factor) (73, 77, 79).The presence or absence of the LA-factor is independent of the state of piliation of gonococci.

12

Alterations in the gonococcal cell surface composition has also been correlated to the concentration of available iron in the environment (48) . Iron starvation induces the appearance in the outer membrane of several high molecular weight proteins. Upon addition of iron these

proteins disappear. These proteins from different strains, that are induced by iron starvation, show a considerable heterogeneity.

Serum resistance, which is found among certain recently isolated strains, is easily lost upon laboratory cultivation. When such a serum sensitive strain was transformed with DNA from a serum-resistant strain, transformants which had recovered serum resistance were found. One of the principal outer membrane proteins of these transformants, showed a reduction in molecular weight. Serum resistance seems thus to be correlated to a specific outer membrane protein (26). Recent gonococcal isolates have also been shown to produce an antiphagocytic capsule. This capsule is found in laboratory cultivated strains of both the piliated and the nonpiliated colonial type (11, 28).

The cell surface structure is: important in determining virulence. These data show that the gonococci possess a considerable capacity to alter the composition of their cell surface, thereby readily adapting to hostile changes in their natural habitat.

13

1:5 Deoxyribonucleic acid contentThe gonococcal chromosome has a molecular weight of

89.8 X 10 (39). This genome size is considerably lessthan the genome size of the Salmonella, Escherichia and Bacillus genera and is only 61% as large as the genome of the closely related Haemophilus genus.

Most gonococcal strains carry a phenotypically cryptic multicopy plasmid of 2.6 x 10^ dalton (45, 50, 59, 69, 81). Plasmid species of this size were isolated from both types Tl and T3 and were digested with restriction endonucleases (15). A comparison of the resulting fragment patterns indicated a complete homology between the plasmids from virulent and avirulent strains. This finding has been confirmed by other authors (10). A detailed physical map of the 2.6 x 10 dalton plasmid indicated the presence of several putative inverted repeats (10). These

inverted repeats have been suggested to play an important role in the observed structural lability of the plasmid (15)/ i.e., the deletion and reappearance of a small segment flanked by inverted repeats (10). The existence of a large plasmid of about 24.5 x 106 dalton in mass was first reported by Stiff 1er e t al_. (69) . These authors found the large plasmid coexisting together with the 2.6 x 10^ dalton plasmid. No correlation between the possession of a particular plasmid and colony morphology or piliation has been demonstrated.

14

p-lactamase producing gonococcal strains were first isolated in 19 76. These isolates were shown to harbour either of two plasmids, with molecular weights of 3.3 x 10^ and

fZ4.4 x 10 dalton (12, 57). It was demonstrated that the p-lactamase produced was plasmid mediated and of TEM-1 type (2, 14). DNA-heteroduplex studies revealed that both gonococcal R-plasmids possess approximately 40% of a transposon, TnA (Tn3), which includes the bla gene (56).TnA is a transposon commonly found on R-factors isolated from Enterobacteriacae. The larger gonococcal resistance plasmid is indistinguishable from a penicillin resistance plasmid that was found in Haemophilus parainfluenzae several years earlier. It has been suggested that such a Haemophilus strain transferred by conjugation this R-factor to gonococci (67). In the case of the smaller gonococcal Rv-factor (3.3 x 10^ dalton) it has been suggested to have originated by a deletion of the 4.4 x 106 dalton plasmid, perhaps during a transformational step.

About 50% of gonococcal strains that carry the 4.4 x 106 dalton plasmid are known to harbour, in addition the24.5 x 10 dalton plasmid. This large plasmid is not found in strains carrying the 3.3 x 10 dalton plasmid. The24.5 x 10 dalton plasmid mediates conjugal transfer of the 4.4 x 10^ dalton R-factor in vitro (12, 57). This plasmid was also suggested to mediate transfer of chromosomal markers by conjugation (58). A rapid spread of the R-factor by means of conjugation to a great

15

number of strains can explain the great variation inauxotrophy and chromosomal antibiotic resistance, which

£has been found among strains carrying the 4.4 x 10 dalton plasmid. In contrast, isolates harbouring the 3.3 x 10^ dalton plasmid show only small variations in auxotrophy indicating that these strains have probably emanated from one single clone (57).

1:6 Transformation in gonococciThree distinct systems for genetic exchange have been described for bacteria, namely, transduction, conjugation, and transformation. Despite considerable efforts no bacteriophages, and thus, no transduction have been described for Neisseria gonorrhoeae. Conjugation was simultaneously reported by several authors to occur in the gonococci (1, 12, 40, 57). The conjugal donors, which carried the 24.5 x 10^ dalton plasmid, were able to

gdonate their 4.4 x 10 dalton R plasmid to penicillin sensitive hosts. Conjugal plasmids in different strains, however, mobilized the resistance plasmid with various efficiency (63). No relationship was found between piliation and the ability to serve as donor or recipient in conjugal transfer (12, 57).

Transformation in gonococci was first demonstrated by Sparling (65). He found that competence for transformation was restricted to the piliated gonococcal variants. These

16

variants were found to be competent during all stages ofgrowth. Competence, however, was maximal in lag and earlylog phases (65). The nonpiliated variants, on the otherhand, are classified as noncompetent with transformation

-7frequencies less than 1 x 10 . The reason for thisdifference in competence between virulent and avirulent colonial variants is hitherto unknown. Pili have been speculated to serve as receptors for DNA during transfor­mation. However, experiments with purified pili and labelled DNA failed to show any binding between pili and DNA (66).

Binding of labelled chromosomal DNA has been found to be nearly identical in piliated and nonpiliated cells.However, DNA was taken up to a DNase resistant state only by the piliated cells (66). This DNA uptake to DNase resistance occurs very rapidly without any detectable lag (89) .

In all transformation systems studied, uptake of DNA is dependent on the physical state of the DNA (4 3) . Double- stranded donor DNA is normally more efficient in transformation than single-stranded. In many systems, nucleases convert the initially double-stranded DNA to single strands. This is apparently not the case in gonococci, where the transforming DNA can be reisolated intact from transformed cells (66).

17

A number of genetic markers have been mapped by transfor­mation and a linkage map representing approximately 3% of the chromosome has been constructed from cotransformation estimates (67). Associated with this region of the chromosome are seven antibiotic resistance loci and three auxotrophic markers.

Neisseria gonorrhoeae has been shown to be transformable by both auxotrophic and resistance markers of heterologous origin (61, 66, 88). However, the transformation efficiencies form a gradient which reflects the degree of genetic relatedness between the donor species and the gonococcal recipient.

13

II. SUMMARY OF THE PRESENT WORK

11:1 Colonial variation in Neisseria gonorrhoeae

Colonial variation in Neisseria gonorrhoeae was suggested by Kellogg already in 1963 to have a genetic basis (37). However, no data has been put forth that supports this hypothesis. I initiated a study on colonial variation by following the kinetics of segregation into different colony types.

When piliated cells were grown in liquid medium, segregation to nonpiliated variants increased markedly after cessation of active growth. Similar segregation kinetics were seen within single, piliated colonies, which were allowed to grow for varying durations. There was not any significant accumulation of nonpiliated types until after 24 hours of incubation. This point in time most likely marks the end of the logarithmic growth phase. The variation in the colony opacity associated (COA) proteins similarly increased in frequency after active growth had ceased.Their increase was, however, less marked than that of the segregation from a piliated to a nonpiliated state.

There was a slightly greater tendency for the loss, rather than the gain of COA proteins. Segregation from a piliated to a nonpiliated state thus occurs within a single colony. Moreover, all the different opacity colour variants can emanate from one single cell. In an attempt

19

to explain the relationship between colonial variation and cessation of growth, my attention was drawn to the marked degree of autolysis occurring in Neisseria gonorrhoeae. In gonococci autolysis is initiated at the end of the logarithmic growth phase. The increase of colonial variation, thereby occurs simultaneously with the release of gonococcal DNA from autolyzed cells. If the released DNA plays any role in colonial variation, the frequency of segregation to other colony types would be expected to be sensitive to DNase treatment. My experiments show that both the segregation to nonpiliation and variation in opacity (COA proteins) are DNase sensitive processes thus demonstrating free DNA to be important in determining colonial variation.

0.6ti : T2

A

<0 .1| 0.6 1.4 <0.1 <0.1 0.4

T4y

T4 T30.6

Figure 1 : In vitro colonial segregation frequencies(per cent of total) of Neisseria gonorrhoeae

20

The overall segregation pattern (Fig 1) is similar in most strains. Nevertheless, two strains appeared, during daily restreaking, to have a more stable colonial morphology (unpublished observations). These two strains both lack the 2.6 x 10^ dalton plasmid, which might implicate this plasmid in colonial variation in Neisseria gonorrhoeae. However, isogenic sets of strains with and without this plasmid are required to settle this question.

II;2 Physico-chemical nature of the cell surface of the

different gonococcal colonial variants In collaboration with a group from the University of Linköping, Sweden, I investigated the effect of pili and the COA proteins, components that are important in determining virulence, on the physico-chemical properties of the gonococcal cell surface. The natural environment

Neisseria gonorrhoeae is affected by several variables. One such factor is pH, which has been shown to affect the composition of the gonococcal cell envelope (18). The effect of the pH of the growth medium on the surface charge of gonococcal variants was assayed by determining the distribution of cells in a two-phase partitioning system upon addition of a positively charged substance. It was found that growth at pH 6.0, compared to pH 7.2, increased the negative surface charge of both piliated and nonpiliated variants. At both pH, piliated variants are slightly more negatively charged than the nonpiliated.

21

At the lower pH, the possession of pili seems to increase the tendency for hydrophobic interactions as assayed by the addition of a hydrophobic substance to the basal system. The COA proteins were found to have no effect on neither the hydrophobicity nor the surface charge of the cell surface. The enhanced hydrophobicity of piliated cells is expected since gonococcal pili are rich in hydrophobic amino acids. These amino acids seem to be concentrated in the amino terminal sequence of thegonococcal pili (3, 84).

We have, furthermore, demonstrated that the cell surface of a recent clinical gonococcal isolate, irrespective of its colony morphology, was more negatively charged and less hydrophobic than a strain which had been maintained by daily passages in the laboratory (Table 2).

In addition to pili and COA proteins, there are various proteins, lipoproteins, and lipopolysaccharides exposed on the gonococcal cell surface (34, 87). Neisseria gonorrhoeae has also been shown to produce a capsule (11,28). Variations in any of these structures and theirdistribution may influence the surface charge and hydrophobicity.

22

Table 2 Comparison of the physico-chemical nature of the cell surface of a laboratory maintained strain and a recent isolate grown at pH 7.2

Change of distribution in, a twD Type of strain Component added phase partitioning system

Tl T2 T3 T4

Laboratory TMA-PEG 4 6 4 3cultivated

P-PEG 30 17 18 28

Recent TMA-PEG 13 8 6 7isolate

P-PEG 11 9 14 18

a) TMA-PEG is a positively charged substance and P-PEG a hydrophobic substance.

b) The figures are calculated as the percental increase of material in the top phase plus the percental decrease of material in the bottom phase compared to the distribution in the basal system. Positive figures reflect a net negative surface charge and an increased liability to hydrophobic interactions, respectively.

23

II;3 The role of the 24.5 x 10^ dalton plasmid0

Cells harbouring both a 4.4 x 10 dalton resistance£plasmid and a 24.5 x 10 dalton plasmid are able to

donate their resistance plasmid by conjugation to a penicillin sensitive recipient. This process, which is dependent on the presence of the 24.5 x 10 dalton plasmid in the donor, occurs independently of the degree of piliation of the donor and the recipient (12, 57). Using nonpiliated variants in such a cross, I found a relatively high frequency of recombinant formation also for chromosomal markers. This finding implied that the24.5 x 10 dalton plasmid might also mediate transfer of chromosomal markers as was suggested by Roberts and Falkow (58). Unlike the findings of these authors, the genetic exchange was sensitive to the action of DNase.The exchange of chromosomal DNA in the 11 mating11 mixture could therefore not occur by conjugation.

The alternative genetic transfer process would be transformation. Transformation, however, has been reported to be inefficient among nonpiliated variants. The process of transfer for chromosomal markers was studied more closely in a constructed strain, Um03, which had obtained the24.5 x 10 dalton plasmid from strain CDC67. The frequency of recombinant formation was compared in crosses betweena nonpiliated strain carrying the 2.6 x 10^ dalton plasmid and strains with and without the conjugative plasmid. It was found that the recombinant formation frequency between nonpiliated T4 variants was enhanced about a hundred-fold

24

in the presence of the 24.5 x 10^ dalton plasmid. The resulting recombinants were all nonpiliated.

When piliated variants were used as either donors or recipients in similar crosses, the majority of recombinants were Tl or T2 colonies and the yield of recombinants was a further ten- to hundred-fold higher. It was concluded that plasmid-mediated genetic exchange was not possible to detect in the presence of this efficient transformation. Further experiments, therefore were confined to nonpiliatedvariants. A two-way genetic exchange experiment, with one

6 6 participant carrying both the 2.6 x 10 and the 24.5 x 10dalton plasmid and the other harbouring only the 2.6 x 10dalton plasmid, was performed. The result revealed thatthe DNA exchange occurred mainly towards the straincarrying the 24.5 x 10^ dalton plasmid. Thus, the presenceof this plasmid conferred increased competence fortransformation. Strains which had spontaneously lost thisplasmid were noncompetent.

The hypothesis that plasmids may be involved in transformation in gonococci has been strengthened by recent transformation experiments in which strains with different plasmid content have been used (Table 3). The plasmid-free strain, KH4318, was transformed for chromosomal markers only with a very low efficiency, irrespective of its colony type.Strain KH7764-45, which carries only the 24.5 x 10 dalton plasmid, was transformed with a higher frequency. However, only piliated variants were transformable. The frequency

25

of recombinant formation in piliated KH7764-45 cells, however, was about forty times lower than that of piliated strains carrying only the 2.6 x 10^ dalton plasmid.

Table 3 Effect of plasmid content on transformationfrequencies

Strain Colonytype

Plasmid content (x 106 dalton)

Frequency of transformation3

KH4318 T2 - 2 X io"8T3 - 5 X io"9

KH7 764-4 5 T1 24.5 5 X 10-6T4 24 .5 1 X

001o 1—1

FAI 71 Tl 2.6 2 X 10 4T4 2.6 1 X io"8

a) Selection was for Str . The frequencies are expressed as number of transformantsper recipient. The mutation frequency

R “8to Str was in all cases less than 10

These data suggest a role for plasmids in maintaining competence for transformation. It should be stressed, however, that additional plasmid-free strains have to be investigated before firm conclusions can be drawn.

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II;4 Restriction modification in gonococci Neisseria gonorrhoeae has earlier been reported to produce two restriction endonucleases, Ngol and NgoII, which are isoschizomers of the Haemophilus endonucleases Haell and Haelll, respectively (8, 9, 55). We have identified a third restriction endonuclease activity in a gonococcal strain. This activity, which was found to recognize the same sequence as SacII, has been denoted EndoR * NgoIII. The gonococcal restriction endonucleases NgoII and NgoIII are not produced by the same strains. In contrast, Ngol is found only together with NgoII (8). The DNA from all gonococcal strains, irrespective of endonuclease production, were resistant to cleavage by Haell (Ngol), Haelll (NgoII), SacII (NgoIII). In addition, gonococcal DNA was found to be poorly cleaved by BamHI, even though no gonococcal strain exhibited an endonuclease activity similar to this enzyme.To demonstrate a possible modification of the corresponding recognition sequences a physical map was constructed for

/rthe gonococcal 4.4 x 10 dalton plasmid and the Tn3 fraction of this plasmid was compared with its known base sequence (24). None of the 7 Haelll (NgoII) sites which were determined to be present on this specific part of Tn3, was cleaved by Haelll. The recognition sequence of Haelll is -GGCC- and the corresponding methylating enzyme has been reported to act at the central cytosine residue. Two BamHI sites have been found on the 4.4 x 106 dalton plasmid isolated from E. coli (L. Mayer, personal communication).One of these BamHI sites must be in the Tn3 part of the plasmid as revealed by sequence data. Both BamHI sites

27

seem to be partially protected as only a minor amount of cleavage products were obtained from the 4.4 x 10^ dalton plasmid when isolated from Neisseria gonorrhoeae. The BamHI recognition sequence -GGATCC- can be protected from cleavage by méthylation of the fifth cytosine residue. It is possible that such a modification prevails in the majority of these sequences in gonococci.

The gonococcal resistance plasmid when isolated from E. coli can be cleaved once by Haell (L. Mayer, personal communication). We found no cleavage using this enzyme when the corresponding plasmid was isolated from Neisseria gonorrhoeae. As Haell and consequently Ngol (-PuGCGCPy-) recognize a sequence containing a combination of GC, the mechanism behind this resistance is most likely also due to a cytosine modification. The same argument holds for the observed resistance to SacII (NgoIII) which recognizes the sequence -CCGCGG-.

Gonococcal DNA of different origin were, irrespective of the plasmid content of the host strain, resistant to the action of Ngol, NgoII and NgoIII. This suggests that these modification systems are chromosomally encoded. Furthermore, there was no apparent correlation between restriction endonuclease production and plasmid content.As the one plasmid free strain studied lacked detectable endonuclease production it cannot be excluded that endonucleases may be plasmid encoded.

28

Gonococcal strains were also shown to contain methylated adenine residues. The presence of a méthylation system for adenines was assayed by means of the sequence -GATC--.A number of restriction endonucleases have been reported to specifically recognize sequences containing -GATC- (55). Two of these are Mbol and Sau3A. The latter cleaves the -GATC- sequence irrespective of adenine méthylation; whereas, the former acts only on the unmethylated sequence. Mbol was found to cleave DNA from three gonococcal strains with a high efficiency. However, DNA from two strains was not cleaved by this enzyme, indicating the presence of methylated adenine. DNA from all five strains was susceptible to cleavage by Sau3A as expected. Interestingly, one of the strains, lacking methylated adenine residues in -GATC-, was a spontaneous fast growing derivative of an adenine methylating strain. Thus, the ability to methylate adenine residues seems to be easily lost and is not a lethal event.

11:5 DNA uptake in Neisseria gonorrhoeae The molecular fate of DNA during and shortly after uptake by competent gonococci is largely unknown. Apparently, gonococci keep the transforming DNA in a double-stranded state (66). The uptake of DNA by gonococci was studied using intact plasmids and end-labelled fragments of well characterized plasmids, which were isolated from Neisseria gonorrhoeae and from Escherichia coli. DNA of the 2.6 x 106 dalton plasmid of gonococci, as has been reported for chromosomal DNA, was taken up more efficiently by piliated

29

than by nonpiliated cells in this system. Nevertheless, the nonpiliated cells were able to take up considerable amount of digested plasmid DNA into a DNase resistant state, However, transformed fragments could be identified by autoradiography only from Tl cells and not from T4 cells. The majority of added fragments were present in the Tl cells. The two smallest fragments (110 bp and 325 bp) of a TaqI digest of the 2.6 x 10 dalton plasmid pJD2 appeared, however, to be absent. In addition, a predominance of the larger of the two Hinfl fragments was seen. These findings indicate a correlation between efficiency uptake and fragment size.

DNA uptake in gonococci was reduced after preincubation with heterologous as well as homologous DNA. The heterologous DNA, pBR322, which was isolated from E. coli, competed, however, less efficiently than the homologous plasmid DNA.

Competent Neisseria gonorrhoeae cells were also found to take up heterologous DNA. Thus, the E. coli plasmids pBR322 and pNU6, a 7.6 kb derivative of the former, were efficiently taken up into a DNase resistant form. Auto­radiography showed that a lysate of the transformed gonococcal cells contained all of the pNU6 fragments which had been added to the cells. These fragments ranged in size from 2,639 bp to 234 bp. When a mixture of homologous and heterologous plasmid fragments were added to competent gonococcal cells, gonococcal fragments were preferentially taken up as assayed by autoradiography.

30

III. GENERAL DISCUSSION AND CONCLUSIONS

Colonial variation in Neisseria gonorrhoeae is a well-known phenomenon which has been shown to reflect changes in cell surface composition and degree of piliation. The different components of the gonococcal cell surface will contribute to its biophysical properties, such as the

degree of hydrophobicity and the charge of the cell surface. Variations in these properties may be of paramount importance for the various types of gonococci-host interactions.The possession of pili, for example, will increase the cell's hydrophobicity at pH 6.0 but not at pH 7.2.

The natural environment of gonococci undergo cyclic changes in pH, the concentration of iron, hormones and proteases, and the presence of secretions or menstrual blood. Each of these variables will add a selection pressure for the gonococcal cell surface type that is most suitable for a particular environment.

Thus, in. vivo conditions enrich for piliated types, while growth in. vitro results in a tendency to segregation towards variants lacking pili. Moreover, in vitro there is a tendency towards a lack of COA proteins. In vivo, however, the degree of opacity is dependent on the physiology of the host. Thus, gonococcal isolates from females show significant variations in opacity depending

31

on the day in the menstrual cycle in which they were isolated (29, 30). Cervical protease activity is known to vary during the menstrual cycle. Thus, the protease sensitive opacity types were isolated during periods with low protease activity and vice versa.

Structural diversity in itself, therefore, seems to be a virulence factor. Available data indicate that this lability of gonococcal surfaces has a genetic basis. Genetic instability may be due to a multitude of events.In general, they all include some recombination step.One such mechanism is the phase variation in Salmonella, which is caused by inversion of a control region (90).In one orientation this region causes the expression of the H2 flagellin and a repressor. This repressor acts on the expression of the alternative flagellin, HI, located in a different region of the Salmonella genome. Thus, this latter flagellin is expressed when the control region is in the inversed orientation. The result of this " flip- flop" mechanism is the alternate expression of the two flagellin structural genes.

Such a mechanism may also explain the variation in gonococcal pili. This variation included two phases, with a particular type of pili produced in opaque variants and a distinct pili found in transparent types (60). Moreover, there is a variation in the degree of piliation, where virulent types are piliated and nonvirulent types are nonpiliated. In addition, variants exist with intermediate

32

degrees of piliation.

The mechanism of COA protein variation seems to be more complicated. This variation includes a variety of surface proteins. Their regulation may be similar to the antigenic variation control in Trypanosoma brucei. The total number of surface variants in this organism is not known but has been suggested to run into the hundreds (27). In one model the basis for the variation is thought to be the trans­location of a promotor between different silent structural genes. An alternative explanation is the translocation of a copy of one or another of the genes specifying the antigenic variation (27). This " casette model11 has been shown to be the mechanism leading to variation in mating types of yeast cells. Yeast cells contain a silent copy of two genes specifying two alternative mating types.The mating type is controlled by insertion of any of these two genes into a specific mating type locus (25, 70).

Whatever the basic mechanisms of the gonococcal cell surface lability are, these processes may be amplified by transformation with autolytically released DNA. The involvement of transformation would explain the marked decrease of colonial variation seen upon DNase treatment. Presumably, changes by a " flip-flop11 mechanism would occur with the same frequency for either orientation. The observed accumulation of specific colonial variants during different environmental conditions would thus likely represent a phenotypical selection.

33

An alternative, but less likely explanation for structural lability, is that during transformation specific incoming DNA may be integrated in either of two orientations, leading to different expression of surface proteins. If so, the variation in the state of piliation would be explained solely by transformation. In contrast, the variation in COA proteins between nonpiliated, noncompetent types which segregate with a frequency as high as the competent variants, cannot be solely caused by transformation. This process, however, is clearly DNase sensitive. Moreover, the presencegof the 24.5 x 10 dalton plasmid which was shown to mediate competence in this strain, markedly enhanced the frequency of segregation from C0A+ to COA . This might be due to enhancement of transformation or alternatively, to a new unknown function mediated by this plasmid.

The DNase treatment might also select for types lacking the COA proteins, as this enzyme often is contaminated by small amounts of proteases. The COA proteins have been reported to be extremely trypsin sensitive and the addition of trypsin will inhibit growth of opaque types (75, 76). The apparent result of DNase treatment is, therefore, an increase of the fraction of variants lacking the COA proteins. This might explain part of the inhibition of segregation seen upon DNase treatment ofT4 variants ' (T4------►T3) . However, the segregation fromT3 to T 4 , which is as DNase sensitive as the reversed process, cannot possibly be explained by such a mechanism.

34

In Neisseria gonorrhoeae there seems to be a relationship between plasmid content and competence for transformation. Piliated variants of a plasmid free strain gave transfor­mation frequencies similar to the spontaneous mutation rate, while piliated variants of strains carrying the

r2.6 X 10 dalton plasmid were highly competent. Furthermore, a strain harbouring only the 24.5 x 10^ dalton plasmid was efficiently transformed when piliated variants were used as recipients. This latter frequency, however, was reduced in comparison to the transformation of strains carrying the 2.6 x 10 dalton plasmid. Nonpiliated variants of one strain, carrying both the 2.6 x 106 and24.5 x 10 dalton plasmids were transformed with a frequency similar to that obtained for a piliated strain harbouring only the larger plasmid. However, all these strains are not isogenic and some differences might therefore be due to strain variations. One piece of evidence, which supports the involvement of plasmidsin transformation is the fact that the loss of the24.5 x 10 dalton plasmid from the nonpiliated strain carrying both plasmids, results in noncompetence.

T1 variants from strains carrying the 2.6 x 10^ dalton

plasmid were found to take up more DNA than T4 variants.T4 cells, however, did take up a certain amount of DNA suggesting that the marked differences in transformation of specific markers is not solely due to a difference in uptake. This indicates that the fate of incoming DNA is not the same in Tl and in T4 cells.

35

It is not known whether Tl and T4 cells differ in their

degree of DNA modification and restriction. Autoradio­graphy, however, failed to show any intracellular intact fragments in T4 cells after uptake. The cell-associated label appeared in this case as a smear at a position expected for degraded DNA. This may indicate the presence of a more efficient DNA degradation system in T4 cells than Tl cells of Neisseria gonorrhoeae. Another possible mechanism for the observed difference may be in some recombinational step.

Piliated as well as nonpiliated variants of a plasmid-free noncompetent strain exhibit a degree of uptake similar to noncompetent variants of strains carrying the 2.6 x 10 dalton plasmid (unpublished observations). This plasmid might therefore encode for functions involved in later steps of gonococcal transformation. In some way these functions or their regulation may interfere with the regulation of pili production, thereby allowing a correlated appearance of competence and pili.

The fact that mainly piliated virulent gonococcal types are competent for transformation, makes it especially interesting to elucidate each step in the transformation process of this organism. If variation in cell surface in vivo also depends on transformation, then clearly transformation in itself may be regarded as a virulence factor. A central question concerning transformation in gonococci is the mechanism by which heterologous DNA is

36

discriminated and homologous DNA recognized. My suggestion is that the gonococcal transformation system, as judged by uptake of plasmid fragments, involves a specificity of DNA uptake, although the distinction between homologous and heterologous DNA is not nearly as complete as in the Haemophilus transformation system. In the latter organism a specific periplasmic protein recognizes and binds a specific DNA sequence, which is probably twelve base pairs

long (7, 62, 71). This sequence, which is present only in homologous DNA, mediates an efficient surface discrimination between homologous DNA and foreign DNA. By comparing the uptake of different DNA fragments the recognition sequence in Haemophilus DNA was estimated to appear every 4.0 kb.

It is possible that gonococcal DNA contain a considerably shorter recognition sequence or that several different regions are recognized and bound. Such regions of low- specific character would be expected to occur also inE. coli DNA, which apparently is taken up by gonococci.

Alternatively, our data may be explained by a specific modification pattern which is recognized by gonococci.Sox et auL. have presented data that indicate the involvement of host restriction modification in gonococcal transfor­mation (64). These authors failed to obtain transformants with a 4.4 X 10 dalton resistance plasmid, which had been isolated from E. coli. When the same plasmid was isolated from Neisseria gonorrhoeae, penicillin resistant transformants were obtained in the gonococcal recipient.

37

Two restriction endonucleases, Ngol and NgoII, have been described in gonococci. In this thesis a third gonococcal endonuclease, NgoIII, is presented. All strains tested, irrespective of their production of specific endonucleases, exhibited a marked resistance to cleavage by Ngol, NgoII, and NgoIII. These restriction enzymes and the corresponding methylating enzymes most likely take part in a gonococcal host restriction modification system. This system may be responsible for the recognition and the degradation of heterologous DNA during transformation. No correlation has been found between restriction endonuclease production and plasmid content. Nor could this modification of cytosine residues and plasmid content be correlated.

Neisseria gonorrhoeae seems to have an unstable dam-like function. Among five strains that were assayed for their ability to have methylated the adenine residue in the DNA sequence -GATC-, three strains were found to lack this function. Moreover, one of the latter strains was a spontaneous mutant derived from an adenine methylating strain. In E. coli, dam mutants have been shown to be spontaneous mutators (44). DNA méthylation seems to be one possible means of discriminating between the parental and the newly synthesized DNA strands. As DNA méthylation is a post-replicative process, newly synthesized DNA chains are not methylated. Replicational errors, which reside in the undermethylated, new strand, would be excised from this strand after discrimination between the two strands on the basis of the extent of méthylation. E. coli

38

dam mutants are hypermutable by the base analogues 5-bromouracil and 2-aminopurine and moderately hyper- mutable by ethylmethanesulphonate and yet these mutants are normally mutable by UV irradiation (16). Apparently, only the correction of base mispairing is affected by the dam mutations. It seems likely that the role of the dam mediated méthylation of adenine residues might be a methylation-instructed DNA repair of mismatched bases, whereby mismatches involving normal DNA bases, base- analogues and some base alkylations can be recognized, excised selectively from the new DNA strand, and replaced by correct bases during the repair synthesis.In agreement with this hypothesis is the finding that transfection of E. coli wild type strains with phage heteroduplex DNA which had only one of the DNA strands methylated, resulted in the preferential loss of genetic markers carried by the unmethylated strand (54). Gonococcal strains lacking adenine méthylation may be mutants deficient in DNA mismatch-correction and may therefore be expected to have an increased mutation frequency.However, there has not been found any evidence for such an increased instability of auxotrophic markers among the méthylation deficient gonococcal strains (unpublished observation). It is possible that variation in the modification of -GATC- and other sequences have implications for pathogenicity of the gonococci. A comparison of modification patterns between strains giving rise to uncomplicated gonorrhoea and those giving rise to dissiminated .infections may therefore be fruitful.

39

In the future a further elucidation of the cell surface lability of Neisseria gonorrhoeae will require new approaches. One obvious approach is to clone the genes for pili and COA proteins in E. coli. After their identification, these clones could be used as probes in blotting experiments with DNA isolated from the different colonial variants. Any variation in DNA structure between the different variants could in this way be elucidated. This approach also seems suitable for develop­ment of a gonococcal vaccine. Good candidates for a vaccine is a composite set of outer membrane components.

40

ACKNOWLEVGUENTS

I am òlneeAeZy gAoteiuZ

to oJUL intend* and colleagues a t the. Ve.paAtme.nt oi UlcAobtoZogy io a many vaZuabZe dt*eu**ton*, keZp and encouAagtng *uppoAt,

In paAtteuLoA, to my *upeAvtòoA Paoì&òAoa S tavan Nonmank ioA hZò tnòptAtng ZnteAeAt and continued * appo At tkAoughout thtò cvoAk and io a htò lohiótling conòeAtò, which gAeatZy òttmuZated my experimental. WOAk,

to Va . John Vavie* io A hi* vaZuabZe *ugge*tion* and encouAagtng ZnteAeAl In the. experiment* and fio a hi* AemaAkabZe ku*tnaLLan *en*e oi humouA, which ioAced me. to escape. Into the. experiment*,

to U*. Chrutlna Fon* io a heA excellent technical a**i*tance and coZZaboAotton,

to the co-authon* oi the papen* ZncZuded tn thi* the*!* oa the iALutiuZ and enjoyable collaboration*,

to U*. Ltòbeth NygåAd and U*. Contea RodAlguez ioA thelA a b tt t ty to pAoduce beautiiul manu*cript* out oi my Zollò y handwAttlng, and * t i l l Aematn my intends,

iinalZy, to Va . Christopher KoAch ior cAltlcaZ Aeadtng oi the manuscript* and *imultaneou*Zy giving me a gAeat Ze**on tn English.

ThZò work wa* *uppoAted by the Swedish Medical Research CounctZ (VnA 4769) and gAantò io A U.V. and Ph.V. *tudent* iAom the Medical Faculty, University oi Umeå.

41

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