' a ‘ : A L U S TV Mi S3i pHH 30 ■ r.'A'rt'1 Jf • j S T R U C T U R E A N D F U N C T IO N O F T H E S U R FA C E L A Y E R O F T H E FISH PA T H O G EN IC B A C TER IU M A m m on as salmonicida by
Rafael A ngel G arduno
B .vS c., E scuela N acional de C iencias B ioldgicas, M&cico, 1980 , . . r , oj . .. }i A D issertation Subm itted in?Partial F u lfilm e n t o f the
‘ ‘ R equirem ents for the D egree o f
,! t( I-?'h i L ;■* S t Of
D O C T O R O F P H IL O SO PH Y
the D epartm ent o f Biochem istry and M icrobiology
W e accept this dissertation as conform ing to the required standard:
D r, W .W . K ay T su p e rv iso r^ e p a rim e p tjO f B iochem istry and M icrobiology)
Dr. A .T, M iilheson, DepartmenuOTlMembor (D epartm ent o f B iochem , and M icrobiol.)
-X 'tN g y 1-..
D r, R .W ; OlaFsdn, D epartm ental M em ber (D epartm ent o f B iochem . and M icrobiol.)
D r. T.W . i3ears()n! D<jnarlmental M em ber (D epartm ent o f B iochem , and M icrobiol.)
-y—y ---- C V " - :---— --- ---D r; L A , H obson, O utside M em ber (---D epartm ent o f B iology)
D r. J. Sm it, External E xam iner (D epartm ent o f M icrobiology, U niversity o f B ritish C olum bia)
© R A F A E L A N G E L G A R D U N O , 1993 U niversity o f V ictoria
A ll rights reserved. D issertation m ay n o t be reproduced in w h o le or in part, by photocopying o r other m eans, w ithout the perm ission o f the author.
Supervisor: Dr. William W. Kay
ABSTRACT
T h e fish pathogenic bacterium Aemmonas salmonicida is the cau sativ e ag en t o f furunculosis in salm onids, a system ic disease that causes im p o rtan t eco n o m ic Josses in salm o n aq u acu ltu re, S ince the paracry stallin e S -lay cr o f Aeromonas salmonicida, k now n as the A -Iaycr, is essential for virulence, the v irulence m e ch an ism s asso ciated w ith this structure w ere studied,
S tru ctu ral studies dem onstrated th a t the A -lay er is flex ib le and p la stic, bein g c a p a b le o f acq u irin g d ifferen t co n fo rm atio n s a n d /o r stru ctu ral p a tte rn s, in w h ich d iv a len t cations play an im portant role,
It w as rigorously dem onstrated (hat the A -lay er acts as an ad h esin , p ro m oting ad h ere n ce to m acrophages, and fish cel) lines. S ince the m acro p h ag e is a p ro fessio n al p h ag o cy te involved in ingesting and destroying bacteria, the ability o f A, salmonicida to re p lic a te in sid e m acro p h ag es w as ex am in ed . A, salmonicida re p lic a te d in sid e m acro p h ag es and eventually destroyed them. This characteristic, together w ith the fact th at A, salmonicida also p en etrated ep ith elial fish ce lls, m ak e it a fa c u lta tiv e ly in tracellu la r, invasive pathogen. T h e A -lay er provided an initial p ro te ctio n a g a in st o x id a tiv e agents, increasing the opportunities for A. salmonicida cells to in d u c e an A - lay e^-in d ep en d en t m echanism in v o lv ed in high resistan ce to o x id a tiv e ag en ts, an d thereby increased survival inside m acrophages,
S tudies w ith in vivo g row n A. salmonicida p ro v id ed fu rth er in s ig h t into the p ath o g en ic p rocess o f furunculosis, and suggested th at the A -lay er plays a cru cial ro le in co lo n iza tio n and penetration o f the host, as w ell as survival in sid e the h o st (early e v e n ts o f the in fe ctio u s p ro cess). H o w ev er, it w as fo u n d th a t in vivo g ro w n A . salmonicida is cap ab le o f ex p ressin g a slim e lay er that sh ield s its en tire su rface an d p ro v id e s full protection against com p lem en t-m ed iated k illin g an d p h ag o cy to sis, th u s relegating the A -layer to a secondary or m inor tole in the later stages o f infection.
T h e results presented have contributed sig n ifican tly to o u r k n o w led g e o f the viru len ce factors o f A, salmonicida, and could be used p ractically in the p rev en tio n o f furunculosis in the salm on aquaeultural industry,
Exam iners;
mwnwViH <Nn:«m
P r, W .W . K ay, S upervisor (P epr^lm erit o f B iochem istry and M icrobiology)
D r, A .T . M atheson. D enartm m rnl. M am h ^' (D epartm ent o f B iochem . and M icrobiol,)
| ;„u ,,...,- T
Dr, R .JV , D iai^o'ivD spSrthibntal M em ber (D epartm ent o f B iochem . anil M icrobial,)
Dr, T 'iW .^ e a iso n , D enartm ental M e r ’ ^D epartm ent o f B iochem . and M icrobiol,)
D r/C , A , H obsop, O utside M em b er (D epartm ent o f Biology)
D r, J. S m it, E x tern al E x a m in er (D ep artm en t o f M icro b io lo g y , U n iv ersity o f B ritish C olum bia)
iv
TABLE OF CONTENTS
A B ST R A C T . ... ii TAB LTD OR CONTENTS... iv LIST OF TA BLES ... ix l i s to r n o
ORES ... .xLIST o r ABBREVIATIONS ... ... ... ...xiv
ACKNOWLEDG EM ENTS... xvii
DEDICATION... xviii G E N E R A L IN T R O D U C T I O N ... 1 P A R A C R Y ST A L L IN E SU R FA C E LA Y ERS O F E U B A C T E R IA ... 1 S -L A Y E R S O F B A C T E R IA L P A T H O G E N S ... ... 4 Aeromanas' bydr op hi la ,,... 6 Aeromonas schubertii ... 8 Bacillus' anthracis ... 8 Bacillus cere us ... 9
Bacillus sphaericus and Bacillus thuringiensis ... ..9
Bacteroides buccae, Bacteroides spp., and o th e r p a th o g e n s associated with p erio d o n titis... 11
Bacteroides nodosus ... 14
Campylobacter fe tu s ... 14
Chlamydia psittaci and Chlamydia trachomatis ... .1 8 Clostridium difficile and other clostridial p a th o g en s... 19
Comamonas acidovorans and oilier p ath o g en s asso ciated w ith suppurative o titis ... .21
M y c o b a e te r iim b o r n ... , ... 2?, Rickettsia species ... ... Treponema pallidum ... ... ... . Aeromonas salmonicida ... ... ... ... O R G A N IZ A T IO N A L PLA N FO R T H IS D ISSE R T A T IO N ... ... .... , . . . 2 3 ...,2(v . , . . 2 7 , . . . 2 8 C H A P T E R I . S T R U C T U R E O E T H E A -L A Y E R O E A erom onas s a lm o n ic id a ... . 2 9 M A T E R IA L S A N D M E T H O D S U SE D IN S T R U C T U R A L S T U D IE S 12 B acterial strain s... 22 G row th c o n d itio n s ... 32 Electron m icroscopy ... 33
C om puter sim u latio n ... 34
Sodium deoxycholatc extraction m ethod lo r A -p ro tein ... 35
T re a tm e n t o f w h o le cells o r purified A -lay ers w ith E D T A o r E G T A , ... 36
T reatm en t o f altered A -layers with divalent c a tio n s ... 36
Im age p ro cessin g ... 36 C H A P T E R I I . A S I N G L E S T R U C T U R A L - T Y P E IN N O R M A L A - L A Y E R S ... 37 IN T R O D U C T IO N ... 37 R E S U L T S A N D D IS C U S S IO N ... 38 C H A P T E R I I I . S T R U C T U R A L P A T T E R N S IN D I V A L E N T C A T I O N - D E P L E T E D A -L A Y E R S , A -L A Y E R S E R O M E N E R G Y S T A R V E D C E L L S O R A - L A Y E R S F R O M M U T A N T S D E F E C T I V E IN A -L A Y E R A S S E M B L Y ... ... ,4 8 IN T R O D U C T IO N ... 48 R E S U L T S ... ,,,..4 9 R eview o f biochem ical studies on A protein subunit In teractio n s. . . 49
E D T A or EG TA altered the A -layer stru ctu re ... 52
A -la y e rs o f ca lc iu m d e p riv e d c e lls ... 52
T h e W h ite D o ts p attern is n o t e x c lu siv e ly associated- w ith lim itation o f di valent c a tio n s, . , , ... 54
VI T h e B ig S quares pattern is form ed by a sin g le m o rp h o lo g ical
U n it I .« I 1 I I . 57
D IS C U S S IO N ..,... 59
C H A P T E R I V . F U N C T IO N S O F T H E A -L A Y E R O F A erom onas s a l m o n i c i d a , , ... ... ... ... ... .
M A TER IA LS A N D M ETH O D S USED IN F U N C T IO N A L S T U D IE S ,. B u f f e r s . . . ... . ... ... Bacterial stra in s. ... ... ... ... G ro w th co n d itio n s an d prep aratio n o f b acterial c e lls ... M urine m acrophages ... Isolation and culture o f rainbow tro u t (Oncorhynchus mykiss) m acrophages ... . ... ... ... Fish cell lin e s ... ... ... ... S taining and counting m e th o d s ... ... ... Electron m ic ro sco p y ... ... ... Survival c u rv e s ... .. ... P u rifie d A -la y e r an d A -p ro tc in ... ,... A -layer reconstitution. , ... , . ... ... C o atin g o f latex b ead s w ith A -la y e r or A -p ro tc in . B acteria-h o stcell association a s s a y s . ... In trap erito n eal ch am b ers used fo r grow th o f A, salmonicida in vivo... ... ... ... S urgery o f rainbow tr o u t., -... ... ... A. salmonicida grow n in vivo ... ... ... C hallenges with scrum and peritoneal f lu i d ... ... . C hallenges with reduced oxygen sp ec ies... ... C hallenges w ith intracellular generators o f su p e ro x id e ... C ytochrom e C reduction a s s a y ... ... ... ... H e m o ly s is a s s a y ... ... ... . 65 ,68 68 68 ...69 .69 ... 70 79 ...8 0 81 83 83 .,...,.8 3 84 84 87 87 89 89 ...89 ...9 0 ... 91 91 C H A P T E R V . A- LA Y E R - M E D I A T E D S P E C I F I C INTERACTION OF Aeromonas salmonicida WITH MURINE
M ACRO PH AG ES ... ... 93
vu
R E S U L T S ,., ... . . , . , . , , 9 4
E ffect o f ■experimental assay conditions on m urine tn 0 s and 4 .
salmonicida ... 94
E ffect o f co m p lex m edia on bacteria-*m 0 in terac tio n s... . 99
A -layer or A -protein coated su rfaces ... ... . 99
A. salmonicida cy to to x ic ity ... ... ... . 102 D IS C U S S IO N ... ... . 102 C H A P T E R VI . A - L A Y E R M E D I A T E D I N T E R A C T I O N O E A e r o m o n a s s a l m o n i c i d a W I T H R A I N B O W T R O U T M A C R O P H A G E S ... . ... 107 IN T R O D U C T IO N ... ... ... ...107 R E S U L T S ..,,... ... ...107
Isolation and culture o f trout m acro p h ag es. ... ...107
R ec o n stitu tio n o f an A -!ayer onto A " , 0 + c e lls ,,,., ... ... . 1 OK A. salmonicida su rface ch an g es and m acro p h ag e asso ciatio n 108 E ffect o f A -layer structural m o d ificatio n s ... ... . . . 1 1 ! E ffect o f hem in coatings on m acrophage a sso c ia tio n ... . I l l C ytopathic effects o f A. salmonicida on trout m a c ro p h a g e s ... . I t 1 D I S C U S S IO N ....,... ...1 14 C H A P T E R V I I . R E S P O N S E O F Aeromonas salmonicida T O O X Y G E N R A D IC A L S . R O L E O F T H E A - L A Y E R ... 120 IN T R O D U C T IO N , ... ,. 120 R E S U L T S ... 121 A ssays on p la te s .. ... 121 A ssays in liquid m e d iu m ... ... ... 125 D IS C U S S IO N ., ... 130 C H A P T E R V I II . I N T R A P E R lT O N E A L C H A M B E R M O D E L F O R i N VIVO G R O W T H O F Aeromonas salmonicida... 137
IN T R O D U C T IO N .,... 137
R E S U L T S A N D D IS C U S S IO N ... 138
R em oved intraperitoneal ch am b ers... 138
S u rv iv al o f A. salmonicida inside in trap erito n eal c h a m b e rs „ ... 140
S u r v iv a l a n d r e p lic a tio n in s id e m a c r o p h a g e s .,.,, ... 143
C H A PTER rx. THE A-LAYER OF Aeromonas CROWN IN VIVO... ... IN T R O D U C T IO N ... . R ESU L T S A N D D IS C U S S IO N , , , , ... ... A -layers o f in vivo grow n A. salmonicida Functional com petence o f the Big Squares pattern salmonicida ... .,.,147 ... 147 ... 147 ...147 ... 148
C H A P T E R X . / A VIVO GROWN A m m o n a s salmonicida.. ...153
IN T R O D U C T IO N ... 153
R E SU L T S A N D D IS C U S S IO N ,., ... . , , ... 155
C H A P T E R X L IS Aeromonas salmonicida AN INVASIVE PA THOGEN?,,,, ... 164 IN T R O D U C T IO N ... 164 R E S U L T S . ... 165 D IS C U S S IO N ... 168 GENERAL DISCUSSION, ... 169 LITERATURE CITED ... 174
LIST OF TABLES
T a b le t . E ffect o f hem in and protoporhyrih IX on the toxicity o f strcptonigrin to A, salmonicida strains A450-1 and A.450-3, as com pared with the w ild type A+ strain A 4 5 0 . ... T a b le 2. B acterio ly tic activity o f trout peritoneal fluid and serum
LIST OF FIGURES
X
F ig u r e 1. S chem atic representation o f the different space sym m etry groups
and arrangem ents found in S -laycrs . . . ... , ...3 F ig u r e 2. T h re e d im e n sio n al reco n stru ctio n o f :U A -la y e r... „ ... .30 F ig u r e 3, Electron m icrographs show ing the two m orphologies
in w hich A -layer sloughs m ay a p p e a r ,... ...3 9 F ig u r e 4. Explanatory m odel o f how A, salmonicida cells
m ay shed their A -lay ers, ..., ... , ... .4 1 F ig u r e 5. H an d ed n ess o f th e A -la y e r lattice a rra n g e m e n t... 42 F ig u r e f». A -layer m orphological types produced by superim position
and differential sta in in g ., ... 43
P ig u r e 7. Lattice constant variation in A -layer slou »hs... 45 F ig u r e 8. M odelling o f the lattice constant variations in
A -laj er slo u g h s --- 45
F ig u r e 9. R egular arrays found in detergent extracted A -layers, o r in sem icrystaliinc sheets form ed upon
concentration o f A -protein on ultrafiltration
m e m b ra n e s . ... 51
F ig u r e 10. A ltered A -layer patterns ... 53 F ig u r e 11. C ell and A -layer m orphologies o f the surface disorganized
m utants A 450-10S and A450-1QSR, as com pared with the w ild
; strain A 4 5 0 ... 55 & 56
Figure 12. C om parison o f the norm al and B ig Squares A -lay er p a tte rn s ... ...58 Figure 13. H ypothetical m odel show ing the proposed structural
rearrangem ents w ithin the A -la y er.,...
Figure 14 , M anufacturing o f laboratory-m ade sieving cu p s... ... ... 72
Figure 15. T o p view representation o f a dissected trout
ju s t before rem oving the head k id n e y ... ... ___ ..74
Figure 16. D istribution o f head kidney cells in a continuous
xi F i g u r e 17. Tips o f the m odified Pasteur pipeis used
in the isolation o f trout m acrophages, ... 76 F i g u r e 18. P reparation o f m acrophage cultures on supported cover,slips . . . 78 F i g u r e 19. Sequential steps in the assem bly o f intvaperuoneal
cham bers using m ieroeentrifuge tubes ... .88
F i g u r e 2 0 . S urvival o f A, salmonicida tit 31%'., ... ... 05 F i g u r e 2 1 . A ssociation o f A * (A450) and A" (A 450-3) bacteria
w ith m acrophages k ep t in PB S, as determ ined by three
different quantitative m ethods and electron m icro sc o p y , ... . , , . . , . . , , , 0 7 F i g u r e 2 2 . Forced association o f A+ (A 450) and A'" (A 450-3)
A. salmonicida w ith m aerw phages in P B S ... ,.0 8 F ig u r e 2 3 . M acrophage association from m ixtures o f A+ (A450)
and A " (A 450-3) strains at different A ” to A + cell ratio s,, , , ... . 08 F i g u r e 2 4 . A ssociation o f A+ (A450) and A"' (A 450-:.,sa lm o n ic id a
w ith m acrophages in tissue culture m edium R P M M 6 4 0 ... 100 F i g u r e 2 5 , R econstruction o f the internalization m echanism o f
A. salmonicida by m urine m acrophages in tissue culture
m edium R P M I-1640... . . . ... . 00
Figure 26. M acrophage association w ith particles or substrata coaled
w ith A -layer or A - p r o te in ....,,.,...,... 101
Figure 2 7 . M orphological changes induced by A. salmonicida upon adherent m acrophages, as detected through scanning
e le c tro n m ic ro sco p y an d lig h t m ic ro s c o p y . ... ..1 0 3
Figure 2 8 . C ongo R ed binding o f the A + reconstituted co-cultured
m ixture, as com pared w ith w ild type A 450, and the separate
reconstitution partners A 4 5 0 -1 and A 4 5 0 -3 .. , . ... ... 109
Figure 2 9 . A ssociation o f trout m acrophages w ith different strains
o f A, salmonicida, and A -layer or A -protein reconstituted
o r la te x b e a d s ... ... ... . 110
Figure 30, M acrophage association o f two different A 450 m utants
w ith structural alterations in their A -iayers ... ... . 112
Figure 31. E ffect o f differen t surface coatings in the m acrophage
asso ciatio n levels o f A +, A 450 and A “ A 450-3
XU
F ig u r e 3 2 . E ffect o f opsonization' o f A salmonicida
upon m acrophage association... . ... 113 F ig u r e 3 3 . E ffect o f A. salmonicida upon trout
m acrophage m o rp h o lo g y ... , ... 115 F ig u r e 3 4 . R ationale o f surface rcconstriuiion with
A -layer sheets from the A450-1 s tr a in ,, . ... 117 F ig u r e 3 5 . T oxicity o f H2O2, streptonigrin, or plum bagin to
different strains o f A salmonicida, as determ ined by the
d isc in h ib itio n a ssa y on n u trie n t a g a r..,. ..., ...122
F ig u r e 3 6 . Effect o f hem in and protoporhyrin IX on the toxicity o f
streptonigrin to A. salmonicida strain A 4 5 0 ... 123 F ig u r e 3 7 . T oxicity o f H2O2 to A. salmonicida cells w ith different surface
properties, as determ ined in liquid phase assays in n utrient b ro th ,. . . 126 F ig u r e 3 8 . Induction, in A. salmonicida, o f a protective, A -layer
in d ep en d en t response a g a in d H2O2... -... <,,», 127 F ig u r e 3 9 . T oxicity o f superoxide to A. salmonicida cells w ith different
surface properties, and induction o f a protective, A -layer independent response, as determ ined in liquid phase assays
in phosphate b u ffer... . . . ... 128 F i g u r e 4 0 . Effect o f different A -layer coatings and grow th conditions
on toxicity o f streptonigrin, H2O2, or superoxide to
A, salmonicida stra in A 4 5 0 ... 129 F ig u r e 4 1 . R eduction o f oxidized cytochrom e C by
x a n th in e /x a n th in e o x id a se g e n era ted s u p e ro x id e ... 131 F ig u r e 4 2 . E ffect o f the presence o f coated or uncoated A + A 4 5 0 cells
o n survival o f the A~ strain A 450-3 to H2O2, streptonigrin, or
superoxide c h a lle n g e s ...,...,... 132 F ig u r e 4 3 . T oxicity o f streptonigrin to A. salmonicida
cells w ith different surface p ro p erties. ... 133 F ig u r e 4 4 . Scantling election m icroscopy o f the internal and external
sides o f a M illipore™ m em brane recovered from an
intraperitoneal cham ber... 139
F i g u r e 4 5 . N egatively stained specim ens show ing the pores
fo rm e d u p o n e x p o su re to p e rito n e al flu id ... 141 F ig u r e 4 6 . G row th o f A. salmonicida A 450 inside peritoneal
xiii
Figure 47. Survival o f different bacterial species after a challenge w ith
fresh peritoneal fluid or fresh serum obtained from trout... 144
Figure 48. Survival o f three A. salmonicida strains inside cultured
head kidney m acrophages isolated from rainbow tro u t. ... 145
Figure 49. A ltered A -layer patterns after in vivo
g row th o f A, salmonicida ... , , ... 149
Figure 50. C haracterization o f A -layets displaying the
B ig S quares pattern in A. salmonicida A 450... 150
Figure 51. R esistance o f in vivo grow n A. salmonicida A450
to different bactericidal challenges... 156
Figure 52. R esistan ce o f in vivo grow n A. salmonicida strains
M T 26 and A450-3 to different bactericidal c h a l l e n g e s , ... 156
Figure 53. E ffects o f in vivo grow th and pre-exposure to fresh
p e rito n e a l flu id on m acro p h ag e a s s o c ia tio n ... . 157
Figure 54. Im m unogold labelling, w ith anti-A*protein, antibody, o f
A. salmonicida A 450 grow n in vivo o r in vitro... 157
Figure 55. E lectron m icrographs o f thin sectioned
A. salmonicida A 450 grow n in vivo o r in vitro , ... ,, 159
Figure 5 6 . N egatively stained cells o f A. salmonicida
A 450-3 grow n in vivo... 159
Figure 57. Survival and m acrophage association o f the
attenuated A. salmonicida strain A 4 5 0 -1OSR
g ro w n in vivo o r in vitro ... ...161
Figure 58. A ssociation of E PC or C H S E fish cell lines w ith
d ifferen t A, salmonicida strain s... ... . 166
Figure 59. S canning electron m icroscopy of EPC and
C H S E cells with adhered bacterial cells o f
the A 450 wild type strain. . — ... 167
Figure 60. T hin section o f an EPC cell show ing several surface-bound
LIST OF ABBREVIATIONS
A TCC A m erican T ype Culture Collection
BSA B ovine scrum aibum in
BS pattern B ig squares pattern
C F U C olony form ing unit
C H S E C hinook salm on em bryo
cm C entim eter
C R C o n g o Red
D'MSO D im ethyl sulfoxide
DOC Sodium deoxycholate
EDDA Ethylcncdiam ine-di(o-hydroxyphenylacetic A cid) EDTA Ethylenediam inc Tetra-aeetic Acid
EGTA E th y len e G l>eol-bis(B -am inoethyl E ther) N ,N ,N ',N ' Tctra-acctic Acid
EM Electron m icroscopy
EPC E pitheliom a p ap ilo su n cyprini
FPA Fixative conUiining form alin and propionic acid in ethanol
FPM F ish peptone m edium
PR units F ree ring units
X gav A verage relative centrifugal field G C co n ten t T otal G uanine-C ytosine content
h H o u i(s)
B B S S H anks balanced salt solution
H E P E S N -[2-H ydroxyethylj piperazinc-N '-[2-ethancsulphonic acid)
IIP L C H igh perform ance liquid chrom atography
1PN Interferon
Im m unoglobulin
IP Intraperitoneal
I PC Intraperitoneal cham ber
Kd D issociation constant
kV K ilovolts
LB broth Luria-Berlani broth
LD50 Lethal dose o f a to x ic o r infectious agent that kills 509; o f a tested animal population
LOS Lipooligosaccharide
LPS Lipopolysaceharide
LTA Lipoteichoic acid
M Abs M onoclonal antibodies
MEM M inim al essential medium
m in, M inute(s)
mM M iilim olar
m0 M acrophage
M .S. 222 3-A m inoben'/oic acid ethyl ester
M W or Mt M olecular w eight
N A D P H N icotinam ide adenine dinucleotide phosphate (in its reduced form)
ng N anogram
nm N anom eter
nM N anom olar
CD O ptical density
OM O uter m em brane
PB M C Peripheral blood m ononuclear cells P B S P hosphate buffered saline
PG Plum bagin
Pi Isoelectric point
pK a -log 10 (acid dissociation constant)
PM N P olym orphonuclear
P M SF P henylm ethylsulfbnyl fluoride
R O S R educed oxygen species
S D S -P A G E Sodium dodecyl sulfate-polyaerylam ide gel electrophoresis
SEM Scanning electron m icroscopy
S N G Streptonigrin
SPA Surface protein antigen (S-layer protein in Rickettsia species)
xvi
SPO Superoxide
TEM Transm ission electron m icroscopy
TE PA B uffer containing T R IS, ED TA , P M SF and sodium azidc
T R IS T dsfhydroxym elhyJjam inom cthane TSA T rypiicase so y agar
TSB Trypticasc soy broth
xvii
ACKNOWLEDGEMENTS
I thank m y su p erv iso r Dr, W .W , K ay for his guidance am,I patience, as well as his g e n e ro sity in p ro v id in g financial su p p o rt as a graduate stu d en t, an d tit attend scientific m eetings.
T h e assistan ce o f G eraldine W on g , Em il Lee, K aren W ithal, A nne A rch er and John W in ch ester, is greatly appreciated.
F in an cial su p p o rt from the U n iv ersity o f V ictoria (through a U n iv ersity o f V ic to ria F e llo w sh ip , P re sid e n t's R ese arc h A w ard and T h e M rs, A n n ie G re sk iw A w a rd ), N S E R C (th ro u g h a P o stg rad u ate S cholarship), and the S cience C ouncil o f B ritish C o lu m b ia (through a G R E A T A w ard) is acknow ledged.
F in ally , I w ant to thank J. N ich o ls for his ad v ise and su p p o rt d u rin g my first y ear a s a g ra d u a te stu d en t, as w ell as B .M . P hipps, J,C . T h o rn to n , J.L . D oran and S.K . C o llin so n fo r being helpful peers.
DEDICATION
A Elizabeth, mi ayuda idbnea
A m is padres por huberm c engcndrado eom o un producto de su am or S obre todo y sobre .lodos, al revelador de m isterios, en especial
el de su presencia en todo y on todos, quo ha hecho y h ace posible todas las cosas en todo lugar. O ne d ay hum anity will have a different approach to the
learning process, T dedicate this piece o f know ledge to the day when the present scientific m ethod
GENERAL INTRODUCTION
T h is d o c to ra l d is s e rta tio n d eals w ith th e stru c tu re and fu n ctio n o f the p arac ry sta llin e su rface la y e r o f the fish pathogen A e tm m a s salmonicida, S everal co m p reh en siv e rev iew s and m onographs on p aracry stallin e surface layers (S -layers) h a v e ap p e a re d re c e n tly (B a u m cisler et al., 1989; B ev e rid g e and G rah am , 1991; E n g elh ard !, 1988; H o v m o ller et ah, 1988a; K oval, 1988; K oval and M u rray , 1986; M e s sn e r an d S lcy tr, 1991 & 1992; P h ip p s, 1988; P um et al., 1989; S ley tr and M essn e r, 1988 & 1989; S leytr et al,, 1988a, 1988b & 1989; S ley tr and Srtra, 1986; S m it, 1986), an d the p ro cee d in g s o f the T hird In tern atio n al W o rk sh o p on S -lay ers (N A T O -A R W , L ondon, O ntario, S eptem ber 27-30, 1,992), are to be published sh o rtly (B ev erid g e a n d K o v al, 1993). C o n seq u en tly , an o th er ex ten siv e p resen tatio n on S- lay ers w ould be redundant. T herefore, this general introduction will d escrib e, briefly, the general characteristics o f cubacterial S-layers, and m ore extensively, an area that has n o t been co v ered in d etail in any o f the ab o v e m entioned review s o r m on o g rap h s, that is th e S -lay ers o f b acterial pathogens, including the m icroorganism that co n stitu tes the su b ject m atter o f this dissertation, Aeromonas salmonicida.
PARACRYSTALLINE SURFACE LAYERS OF EUBACTERIA
H o u w in k (1 9 5 3 ) w as the first to rep o rt the ex isten ce o f an S -layer, T his w as d etected by electro n m icroscopy o f cell w all fragm ents p repared by m etal sh adow ing (u n fo rtu n ately these lay ers are o n ly detected by electron m icro sco p y ). S in ce then, S- layers have been reported in an increasing num ber o f bacterial species. T h e m ost recen t list o f eu b a c te ria po ssessin g S -layers (M essner an d Sleytr, 1992) in clu d es m ore than
200 sp e c ie s an d stra in s, co m p risin g n early e v e ry eu b a c te riu l ta x o n o m ic g ro u p , A lth o u g h th ese m acrom olecular surface structures have received different nam es, a t the S eco n d In te rn a tio n a l W o rk sh o p on S u rface L ayers, held in V ienna in 1987, it w as ag reed to use the term “ S -iay er” to co n n o te “ tw o -d im en sio n al cry stallin e array s o f p ro tein aceo u s su b u n its form ing surface layers on prokaryotic cells” . In this co n tex t S
2 layers sh o u ld be distinguished from oth er surface layers describ ed in p rokaryotes; i.e. cap su les, external slim es o r glycocaliccs, and sheaths (B ev erid g e and G raham , 1991). Interestingly, m an y S -lay er proteins constitute exam ples o f p ro k ary o tic g ly co p ro tein s (M essn er and S leytr. 1991), for m any years thought not to ex ist in prokaryotes.
S-layers arc tw o-dim ensional, highly organized protein m uH im ers d isp o sed as sin g le m o n o lay ers, or as n atu rally su p erim p o sed m u ltilay ers, th a t c o v e r th e ersthc su rfac e o f those b acteria w hich possess them , W ith few ex cep tio n s, each S -’a y cr is co m p o sed (if a single ty p e o f protein o r glycoprotein subunit. T h erefo re, w hen m u lti- S -lay ers are p rese n t on th e sam e bacterium , each has a d iffere n t protein co m p o sitio n (A ustin and M urray, 1990; K ista n d M urray, 1984; T suboi etal, 1982).
S -layer su b u n its are acidic proteins with a m ass ran g e o f 30 to 220 k D a, high co n ten t o f h y d rophobic am ino acid s (40-50% on av erag e), and a very low c o n te n t o f m e th io n in e and cy stein e. S tru ctu rally , S -lay er su b u n its in e u b a c te ria are b ilo b ed p ro tein s, form ed by a heav ier (m ajor) and a lighter (m inor) do m ain co n n ected b y an arm (F ig . 1), H eav ier d o m ain s co n v en e around a p rim ary sy m m e try axis to form m assiv e co res, and the lig h ter d o m ain s p ro v id e c o n n e c tiv ity by in te ra c tin g a t a secondary sym m etry axis. Prim ary and secondary axes m ay be of the sam e o r differen t sy m m e try , i.e. 2-fold, 3 -fo ld , 4 -fo ld o r 6-fold sy m m etry , and reg u la rly d istrib u te d w ithin arrays o f p ! , p2, p3, p4 o r p6 sp ace sym m etry (S ax to n an d B au m eister, 1986) (Fig. 1), T he c u rre n t u n d erstan d in g o f the o rg an izatio n o f S -lay e rs in eu b ac teria, d efin es the su b u n it-su b u n it and the subunit-w all in teractio n s as th e m eans o f S -lay er in teg rity (B au m eister et al,, 1989; K oval, 1988; S le y tr and M essn e r, 1992; S m it, 1986). S ubunit-subunit interactions determ ine the form ation o f the paracrystalline array w hile in teractio n s w ith th e cell w all provide anchorage o f the asse m b led array to the bacterial cell,
Purified S -layer subunits have show n the ability to spontaneously self-assem b le into array s identical to th o se observed on cells, su g g estin g th at array fo rm atio n is an entropy-driven, process, and that subunits possess a lt the info rm atio n necessary to form lattices o f d efin ed stru ctu re (S ley tr and M essn er 1989). P u rified S -lay er su b u n its or layers assem bled in vitro also have the ability to reattach onto cell surfaces devoid o f S- la y e rs. T h is re c o n s titu tio n p ro ce ss is u su ally sp e c ie s a n d /o r stra in s p e c ific , d em o n stratin g th e im portance and sp ecificity o f su b u n it-w all in teractio n s. S u b u n it- su b u n it and su b u n it-w all in teractio n s are gen erally o f a d iffere n t n atu re (A u stin and
Minor domain Ann
IMajor domain
S-layer protein
f ’IG U R E 1. Schem atic representation o f the different space symmetry groups and arrangements found in S-layers, The symmetry axes, around which the S-layer protein major ( M ) , or m inor ((',)
domains convent, are shown as geometric figures filled in black: ellipse = 2- fold symmetry axis, triangle * 3-fold symmetry axis, square * 4-fold symmetry axis, hexagon - 6*fold symmetry axis. P i, P2, P3, P4, and P6 designate the space symmetry groups, and the nomenclature used for (he different lattice arrangements is that proposed by Saxton and Baumeister ( & 86). Other symmetry centres within the lattice are-not shown,
4 M u rray , 1990; B aum eister <'t. a l, 1982; D o ran e: al., 1987; K oval and M u rray , 1983; N o rm u t and M urray, 1967; S ley tr and G lau ert, 1976; S leytr and P lo h b e rg e r, 1980), and arc m ediated by non-eovalent bonds (hydrophobic and electrostatic interactions), as In ferred from the m ethods required- to iso la te in tact S -layers o r to so lu b iliz e S -lay er pro tein s (KOnig and S tcttcr, 1986; K oval an d M urray, 1984; M essn er & S leytr, 1992), T h e se m ethods usually involve th e use o f ch ao lro p es, detergents, lo w io n ic stren g th buffers or deionized w ater, extrem e pH buffers, or divalent catio n chelators.
S -layers are clearly im portant su p ram o lecu lar assem blies, acco u n tin g fo r th eir u b iq u ity am o n g p rokaryotes. W h en p resen t, they co m p rise up to 2 0 % o f the total cellu la r protein (im posing an expensive burden upon the cell econom y), and it has been estim ated that they rep resen t the m ost ab u n d an t prokaryotic proteins in the b io sp h ere (M essn er and Sleytr, 1991 & 1992), In m o st cases how ever, their e x a c t b io lo g ical fu n c tio n s h av e n o t y e t been assig n ed . In d eed , in c o n tra s t to th e ir s tru c tu ra l ch ara cterizatio n , there is a severe lack o f in fo rm atio n regarding the fu n ctio n s o f these unique structures, O w ing to their strategic surface location, and to the fact that S-layers rep resen t ttpen netw orks o f charged proteins w ith defined pores, the follow ing potential fu n ctio n s have been proposed: i) pro tectiv e coats, ii) m o lecu lar siev es, iii) m o lecu la r trap s an d /o r io n ic ex ch a n g ers; iv ) ad h csin s for su rface re c o g n itio n ; v) c e ll sh ap e d eterm in an ts, A lth o u g h som e o f these fu n ctio n s h av e now been co n firm ed in so m e eu b acteria (M essn er and Sleytr, 1992), th e ev id en ce is still frag m en tary o r a b sen t in m o st cases.
S-LAYERS OF BACTERIAL PATHOGENS
The interaction o f bacterial pathogens w ith th e ir h o st is m ainly d eterm in ed by m aero m o lecu lar surface structures (B row n and W illiam s, 1985, D oyle and S onnenfeld, 1989, F ish er, 1989, S m ith , 1977, W illia m s, 1988). T h e p a th o g e n ic p ro c e ss o f infectious d iseases has been dissected in th e follow ing steps: co lo n izatio n , p en etratio n , m u ltip lica tio n , ev asio n o f host defense m e ch an ism s and dam ag e to th e host. In all th ese steps (w ith the possible exception o f m ultiplication in th e host an d d am ag e) the b a cteria l s u rfa c e plays a piv o tal role. I t is the c e ll su rfa c e , th ro u g h sp e c ia liz e d m ac ro m o le c u lar structures know n as a d h em s, w hich serves as an an ch o rin g p o in t for path o g en ic bacteria, In turn, these adhcsins m ay determ ine h o st and tissu e specificity,
5 i.e. th e ability o f a b acterial pathogen to p ro d u ce disease in so m e an im al sp ecies in p referen ce to o th ers, and the ability to co lo n ize (w ithin a given host) som e specific tissue in preference to others, It is the cell surface, through its sp ecialized structures, w hich determ ines w hether certain pathogens w ill be invasive, i.e. able to penetrate host cells o r som e physical barriers, to becom e established in deeper layers o r tissues o f the host. S u rface co m p o n en ts co n trib u te to m ultiplication in the h o st by help in g bacteria ac q u ire e sse n tia l n u trien ts. O n e o f the b est docum ented cases is th eir role in iron aquisition, e ith e r through specific surface receptors for siderophores o r th ro u g h direct b in d in g o f h o st iron-containing m olecules. It is also the cell surface w hich serv es as a site o f im m unological recognition, accounting for the im portance o(surface antigens in v accin e design. It is by m odifying their cell surface (through m echanism s o f antigenic and phase variation) th at som e bacterial pathogens ev ad e im m unological recognition. As w ell, it is th e cell su rface w hich serves as the targ et o f m any o f th e host defense m echanism s, and it is by having tough specialized surfaces that so m e pathogen,,, evade th e se m e ch a n ism s. F o r in sta n ce, so m e su rfa c e stru c tu re s (e.g , p o ly sa c c h a rid e c ap su les, lo n g O p o ly s a c c h a rid e ch ain s and S -layers) in h ib it co m p lem e n t-m e d ia te d k il lin g , p h a g o c y to s is a n d /o r in tr a c e llu la r d ig e s tio n a f te r p h a g o c y to s is . L ip o p o ly sacch a rid e (L PS) from gram -n eg ativ e bacteria, and lip o teich o ie acid s (L'l'A ) from g ra m -p o sitiv e b acteria, also have been im plicated in m o d u la tio n o f the h o st im m u n e resp o n se. Finally, surface com ponents o f bacterial pathogens m ay cau se host d am ag e evoking im m unopathological reactions (e.g. im m unopathology associated with surface antigens o f Treponema pallidum or Mycobacterium species).
T he su rface location o f S -layers, in m any instances as th e o u term o st stru ctu re on bacterial pathogens, m eans that they serve as the interface betw een the pathogen and its h o s t and m ay significantly influence the outcom e o f a host-parasite relationship, S- la y ers m u st n a tu ra lly com e in clo se co n tact w ith cells, flu id s, m e m b ra n es an d /o r b a se m e n t p ro te in su rfaces o f the host, and th erefo re m ediate sev eral o f the actio n s described above. In the follow ing section a survey o f cases o f bacterial path o g en s wi th S -lay ers (ordered alphabetically by genus and species) is presented. M y intention is to em p h asize the know n virulence functions o f S-layers but, unfortunately, only structural descriptions are available in the m ajority of cases.
A e r o m o n a s h y d r o p h ila
A ccording to the B crg ey ’s M anual o f S ystem atic B acteriology (P o p o ff, 1,984), the g en u s Aeromonas belongs to the fam ily Vibrionaceae and in clu d es tw o g ro u p s sep arated on the basis o f o p tim al grow th tem p eratu re and m o tility . O n e g ro u p is form ed by p sy ch ro p h ilic, n o n -m o tile acro m o n ad s, b elo n g in g to a sin g le sp ecies, A. salmonicida, w ith three subspecies, A. salmonicida subsp. salmonicida, A. salmonicida su b sp . achromogenes, and A. salmonicida subsp. masoucida. T h e sec o n d g ro u p co n sists o f m eso p h ilic and m otile bacteria, including three sp ecies, A. hydrophila, A. caviae and A. sohria. A lthough the taxonom y o f m esophilic aero m o n ad s has ch an g ed significantly from P o p o ff s schem e, the psychrophilic group has been m o re stable. T o date, it has been proposed to m ove the acrom onads out from the Vibrionaceae to form a new fam ily, the Aeromonadaceae (C olw ell, e ta l, 1986); the taxonom y o f the n o w 11 recognized sp ecies o f m esophilic acrom onads is still n o t reso lv ed (M artfn ez-M u rcia et at,, 1992); an d a new su b sp ecies o f A. salmonicida h as been pro p o sed (A u stin et a l, 1989; B eilan d and T ru st, 1 9 8 8 ) lo in clu d e the so -c a lle d “ a ty p ic a l” stra in s. A . hydrophila co m p rises g ram -n eg ativ e straig h t and sh o rt rods, m o tile by a sin g le p o lar liag ellu m in liq u id m edium an d som etim es p eritrich o u s flag ella on so lid m edia. Its o p tim al g ro w th tem p eratu re is 28°C . Its nam e m eans “ w a te r-lo v in g ” sin c e th is organism is co m m o n ly found in fresh w ater and sew age (P opoff, 1984).
In 1980, M ittal et al, (1980), rep o rted a p a rticu la r g ro u p o f A. hydrophila strain s that w ere highly v iru len t to salm onids. A t th a t tim e, A. hydrophila had been regarded only as an op p o rtu n istic pathogen (B oulanger e ta l, 1977; De F ig u eired o and P lum b, 1977), residing free in the environm ent (H azen e ta l, 1978; K aper etal., 1981) o*’ in the d ig e stiv e tra ct o f salm o n id s (T ru st and S p arro w , 1974; C a h ill, 1990b and references therein). H ow ever, the high virulence traits o f this particular g ro u p indicated that som e A, hydrophila iso lates m ay behave as true prim ary pathogens. P ath o g en ic A. hydrophila c a u se h em o rrh ag ic sep ticem ic in fectio n s in fish , and g a stro in te stin al or sy ste m ic in fe c tio n s in o th e r an im al sp ecies in c lu d in g a m p h ib ia n s, re p tile s, and m am m als. In te re stin g ly , certain A. hydrophila strain s iso la ted from h u m a n s also d isp lay this high virulence phenotype (D ooley, etal., 1985; Jan d a etal., 1987), v/hich sig n ific a n tly c o rre la te s w ith p ro d u ctio n o f in v a siv e in fe c tio n s (b a c te re m ia and perito n itis) (Ja n d a etal., 1987). T he highly v iru len t iso lates (eith er from salm o n id s, m a m m a ls o r h u m a n s) p o sse ss O -p o ly s a c c h trid e c h a in s o f h o m o g e n e o u s len g th (D o o ley , et at., 1985) th a t carry a th erm o stab le se ro g ro u p -sp e cific ep ito p e , 0 :1 1
7 (S akazaki, 1987), in turn associated with the presence o f a pnracrystalline surface layer (D o o ley and T ru st, 1988). T he S -laycr o f the highly v iru len t A. hydrophila strain s (particularly T F 7 ) has been well characterized, In it" relation w ith the 0 ; 11 specific 0* p o ly sa c c h a rid e ch ain it w as sh o w n th a t S -lay e r an c h o ra g e is 0-p o ly sa c c h a rid e - d ep en d en t and that a m inim um L P S oligosaccharide size is required for an ch o rin g the S -la y e r to the cell su rfa ce (D o o ley and T rust, 1988; K okka et al,, 1990), A ll the m em bers o f the high virulence group possess a sim ilar te tra g o n a l^ arrayed layer w ith a lattice co n stan t o f ab o u t 12 an (M urray et cd„ 1988). T h e 3-D structure o f this array has b een reso lv ed (A l-K arad ag h i etal,, 1988) sh o w in g th a t the S -lay er su b u n its are b ilo b ed p ro tein s g ro u p ed around two d ifferen t 4 -lb ld sy m m etry axes w ith an MqOq arran g em en t (refer to Fig, 1) and a thickness o f 5.6 nm. D ooley et al,, (1988; D ooley and T ru st 1988) carried o u t the biochem ical characterization o f the S -layer subunit from strain T F 7, T h is is a 52 kD a acid ic protein, w ith 41% h y d ro p h o b ic am in o acid s, a sin g le iso electric form (p i 4.6), and no cysteine. V ery sim ilar ch aracteristics w ;rc also fo u n d fo r the S -la y e r p ro tein o f a strain (A H -3 4 2 ) in v o lv ed in a case o f hum an b acteraem 'a (K o k k a etal,, 1992b). A lthough hydrophobic in itself, the S -lay er protein d o es n o t c o n fe r h y d ro p n o b icity to A, hydrophila cells w hen p resen t as an in tact S- layer. T h e S -lay e r p ro te in is co m p o sed by 44% b eta-sh eet, 19% alp h a-h clix , 12% beta-tu rn , and 25% aperiodic structure, T he N -tcrm inal am ino acid seq u en ce (first 30 residues) o f the m atu re protein did not show any sequence sim ilarity w ith oth er S -lay er pro tein s (D ooley and T rust, 1988; K okka etal,, 1992b).
E x p lo rin g the an tig en ic related n css o f the S-Iayers asso ciated w ith the ();! I sero ty p e, it w as fo u n d th at som e S -layer proteins w ere a n tig e n ic a l'y d iv erse ow ing lo differences in their prim ary sequence. D ue to the com plexity o f this genus, this is not a su rp risin g finding, and in relation to pathogenesis, it m ay rep resen t an extra strategy o f a n tig e n ic v ariatio n o f S -lay er-p o ssessin g A, hydrophila strain s (K o str/.y n sk a el al., 1992). E stab lish m en t o f the functions for the S -laycr o f path o g en ic A. hydrophila has not b een an easy task, T h e stro n g est d irect evidence o f its role in p athogenesis co m es from rece n t experim ents reported by K okka et al, (1992b). S -layer negative (S ')strain s w ere injected intraperitoneally, together w ith purified S -layer protein, in S w iss-W ebster m ice, In terestin g ly the LD$r o f S" strains w as reduced 3 0 -70 fold, su g g estin g that the S -lay e r p ro tein m ay p lay an im p o rtan t role in p ath o g en esis. T he m echanism o f this enhancing effec t upon the ability to produce disease is not y et know n. H ow ever, it w as p ro p o sed that the S -lay e r su b u n its reconstituted an S -lay er on the su rface o f S* cells,
8
co n ferrin g to them so m e trait(s) necessary fo r sy stem ic d issem in atio n (K o k k a e ta l, 1992b), It is d isa p p o in tin g that, alth o u g h c le a rly im p lic ated in v iru le n c e and p athogenesis (M ittal et al.,, 1980; Janda et al,, 1987: Ford an d T hune, 1991; K o k k a et a l, 1992b), a sp e cific fu n ctio n fo r this w ell ch ara cte riz ed S -lay e r o f v iru le n t A. hydrophila, has not as y et been unequivocally dem onstrated (K okka et a l, 1991).
A e r o m o n a s s c h u b e r tii
T his is one o f the 12 recognized species o f the genus Aeromonas and belongs to the g ro u p o f m e so p h ilic m o tile ae ro m o n ad s (M a rtln e z -M u rc ia et al,, 1992 an d referen ces therein). A lthough d ifferen t species o f aero m o n ad s p o ssessin g the 0 :1 1 sero ty p e also possess an S -lay er (e.g. A. veronii),A. schubertii is o f p articu la r in terest b ecau se it cau ses sy stem ic o r w ound infections. S ig n ifican tly , a lth o u g h o n ly a few strains have bet n isolated, none o f them w ere isolated from the gastrointestinal tra ct and no an im al o r en v iro n m en tal isolates are know n to exist. K o k k a et al, (1 992a) stu d ied the 11 know n strain s o f A. schubertii to d eterm in e th eir stru c tu ral and p a th o g en ic pro p erties, A ll six strain s b elo n g in g to the 0 :1 1 sero g ro u p (b u t n o n e o f th e strain s co n tain in g L P S w ith O -ch ain s o f heterogeneous length) p o ssessed a 55 k D a su rface la y e r pro tein . O n e, o u t o f th ree strain s w ith tru n c ated O -p o ly sa c c h a rid e ch ain s, secreted the S -lay er p ro tein into th e m edium , su g g estin g th at, as d escrib e d fo r A . hydrophila, an ch o rin g o f the S -la y c r to the cell su rfac e is L P S -d e p e n d e n t a n d a m inim um len g th o f the O -p o ly sacch arid e ch ain is required fo r co m p eten t an ch o rin g (K o k k a et al. 1992a). S tran g ely , these authors d id n o t re p o rt any u ltrastru ctu ra l o r functional characterization of the S-layer.
B a c illu s a n th ra c is
T h e m em bers o f the g enus Bacillus are distinguished fro m o th er g ram -p o sitiv e rods b y th e ir stric t o r facu ltativ e aero b ic nature, and their ab ility to sp o ru la te and p ro d u ce catalase fS lep eck y and H em phill, 1992). B. anthracis, the cau sativ e ag en t o f the fatal disease anthrax, p o ssesses tw o w ell reco g n ized v iru len ce factors: a p o ly-D - glutam ic acid cap su le w hich inhibits phagocytosis (G reen etal., 1985) and a tripartite toxin (L eppla, 1988). T h e p resen ce o f a lin ear S -lay cr in th is o rg an ism Was early reco g n ized in freeze-etch ed rep licas (H o lt and L eadbetter, 1969) w h ere the S -lay er ap p eared to be form ed by strands o f globular particles o f 6-8 nm . S eparation betw een stran d s an d b etw een p articles w as 7 -1 0 nm . A n aly sis o f a 2-D p ro je ctio n o f the
y
n eg a tiv ely sta in e d S -lay er sh o w ed th a t it is o rg an ized in a p i la ttice (D o y le and S onnenfeld, 1989). A lthough u is possible that tw o proteins designated as “cxtrnetablo an tig en s” E A -I and E A -II (closely associated w ith the pep tid o g ly can layerl are the S* la y er su b u n its (D o y le and S onnenfeld, 1989), th e ir id en tity has n o t been clarifie d , n eith er their function discerned.
B a c illu s c e re u s
B. cereus has been reco g n ized as an a g e n t o f food p o iso n in g sin c e 1955, c a u sin g tru e in to x ic a tio n s ra th e r than in fe ctio n s. H o w ev er, B cereus has been
im p lic ated in m u ltip le cases o f a variety o f infections (F arrar and R eb o li, 1992), and acco rd in g to D av cy and T au b er (1987) th is is one o f the m o st destructive organism s to in fe c t the eye. Since B, cereus produces potent exotoxins (responsible for m uch of the p ath o g en esis asso ciated w ith this organism ) em phasis has been placed on the study o f su ch toxins. T h u s the functions associated with its S-layer have not been characterized a t all, and ev en stru ctu rally , very little is know n ab o u t the S -lay cr o f B. cereus, H o lt an d L ead b etter (1969) described it as a “globular lay er” , sin ce in freeze-etched replicas it ap p eared as form ed by g lo b u lar units o f 6-7 nm in size. N eg ativ e staining revealed m o re cle arly a tetrag o n al array w ith a lattice sp acin g o f ap p ro x im ately 10 nm , first described by E llar and Lundgren (1967) on the A T C C strain 4342.
B a c illu s sp h a e r ic u s a n d B a cillu s th u rin g ien sis
In the co u rse o f sporulation, so m e strains o f Bacillus sy n th esize a parasporal in c lu sio n o r “c ry sta l” , w hich m ay co n tain pro tein s toxic for la rv ae o f a v ariety o f in sects. T h e tw o m ore p ro m in en t sp ecies o f these en to m o p ath o g e n ic bacteria are B, sphaericus a n d '# . thuringiensis. C om m ercial products (bioinsecticides), prepared from c u ltu re s o f th ese m otile Bacillus species, are available and they are ex p ected to gain p o p u larity s in c e they do not h av e m ost o f the en v iro n m en tally d eleterio u s effects o f sy n th etic in secticid es (Ignoffo and A nderson, 1979; Stably etal., 1992). In terestin g ly , both sp ecies h av e S-layers.
T h e S -lay e r o f B, thuringiensis w as d escrib ed recen tly b y L u ck ev ieh and B ev erid g e (1989). T he three strains that they stu d ied had stru ctu rally sim ilar S -lay ers c o m p o se d o f a d elicate o b liq u e array. O ptical d iffractio n o f n e g a tiv e ly stain e d sp ecim en s sh o w e d that the m ajo r lattice lines w ere spaced ab o u t 8.5 nm . S eco n d ary lattice lines, ru nning at 73° w ith respect to the m ajor lines, w ere separated 7 .2 nm. T h e
10 la y e r w as 9 nm th ick in thin sectio n s. S h ad o w ed p re p ara tio n s o f sh ed S -la y e r fragm ents sh o w ed that the layer has an external sm ooth face and a rough face facing the cell w all. T h e p u rified S -lay er protein w as hydro p h o b ic, an d acid ic (p i 5) w ith a m olecular w e ig h t of 91,400, n o t glycosylated, and did not contain cysteine, m ethionine and tryptophan. Its N -term inal am ino acid sequence w as d eterm ined but no seq u en ce sim ilarity w as found w ith any o th er S -lay er protein. In creasin g am o u n ts o f S -lay e r protein w ere found as cultures aged, and stationary p h ase cells shed larg e am o u n ts o f S -lay cr fragm ents, N o functional characterization w as attem pted.
in c o n tra st to th e S-layer o f B. thuringiensis, the S -lay er o f B. sphaericus w as rep o rted m u ch earlier (H o lt and L cadbettcr, 1969). H ow ever, it w as n o t until 1983 w hen it w as d iscovered that the cn tom opathogenic strains o f B. sphaericus had an S- lay er d ifferen t from th a t o f non-pathogcnic strains (W ord et al,, 1983). T h e S -lay e r o f n o n -p ath o g o n ic strains consists o f a tetragonal array w ith a lattice co n sta n t o f 13-13.5 nm , form ed b y a — 150 k D a protein. Interestingly, S -lay er n eg ativ e m u tan ts h av e not b een iso la te d , su g g e stin g an in d isp e n sa b le ro le fo r th is S -la y e r, a lso sh o w n to co n stitu te a b acterio p h ag e recep to r (H ow ard and T ip p er, 1973). T h e S -lay e rs from n o n -p ath o g en ic B, sphaericus strains have been used to study th e d y n am ic p ro cess o f S -lay e r assem b ly d u rin g grow th (Sleytr an d G lauert, 1975; H o w ard et a l, 1982), to dem onstrate th e bilobal nature o f the S-layer subunit and define the dom ains and bonds involved in array form ation as w ell as anchorage to the cell w all (H astie an d B rin to n , 1979a; H o w ard and T ip p er, 1973; L epault etal,, 1986), to stu d y the sp ecificity o f S- la y cr reco n stitu tio n (HastiC and B rinton, 1979b), and the v ariatio n in fin e stru ctu ral detail betw een air-dried negatively stained and unstained fro zen -h y d rated sp ecim en s (L e p a u lta n d Pitt, 1984; L epault etal., 1986).
AH th e p ath o g en ic strain s o f B. sphaericus p o ssess o b liq u e S -lay ers w ith a sp acin g o f 5 nm and fa in t secondary linearity at ab o u t 27° (L ew is et al., 1987). T h ese co v er the en tire cell surface, and have the ability to reassem ble in vitro. In spite o f their stru ctu ral h o m o g en eity , it w as found that S -layer proteins o f p ath o g en ic strain s w ere an iig en ically heterogeneous. T h e re was a p erfect correlation betw een an tib o d y typing and the type o f peptide m ap obtained upon trypsin digestion, su g g estin g th a t an tig en ic variation w as due lo differences in prim ary am ino acid sequence. H ow ever, all S -lay er p ro tein s sh ared m any b io ch em ical characteristics, all w ere g ly co sy lated , had sim ila r am in o acid co m p o sitio n s, m olecular m asses (133-155 kD a) and isoelectric points (4,6- 4 .9 ). In tere stin g ly , sin c e the S -lay ers o f pathogenic strain s o f B. sphaericus w ere
lin e a r (as th o se o f B. thuringiensis) w h ereas n o n -p a th o g e n ie s tra in s p o sse ssed te trag o n al array s, a p o ssib le relatio n sh ip betw een S -lay e r and p a th o g e n icity w as co n sid e red an d further supported by the fact that a p roteolytic deg rad atio n p ro d u ct o f the S -lay er protein o f strain 2362 appeared to be to x ic to insects. P rep aratio n s o f the larv icid a l crystal show ed that it w as form ed by four autigenically related proteins, 125, 110,, 63 and 43 k D a (B aum ann et a l, 1985). E x c itin g resu lts sh o w ed th a t upon com pletion o f the exponential phase, and initiation o f the sporulation process, a gradual decrease o f the ab u n d an t 125 kD a protein w as accom panied by a proportional increase o f th e 110 k D a protein. S ince the latter proved lo be toxic to m osquito larvae, but not as to x ic as the 43 k D a protein, it w as proposed that the 110 and the 43 k D a parasporal c ry s ta l c o m p o n en ts rep rese n ted su b se q u en t d eg rad atio n p ro d u cts o f the 125 kD a protoxin, each having increased toxicity against insect larvae (B roadw el! and B aum ann, 1986). O nce the gene encoding the 125 k D a protein w as cloned and seq u en ced , it w as fo u n d that the 125 kD a w as the precursor o f the 122 kD a S -layer protein o f strain 2362, an d th a t the 125, 122 (S -lay er p ro tein ) and 110 kD a p ro te in s sh a red the sa m e IM- te rm in u s a m in o a c id se q u en ce (B o w d itch et a l, 1989). H o w e v er, it w as also dem onstrated that no relationship existed between this group o f proteins and the 43 kD a to x in , and th e p ro p o sed relatio n sh ip betw een S -lay er and to x icity beg an to collapse. N o w it is k n o w n th a t no relatio n sh ip ex ists betw een S -lay er and p ath o g en icity o f IS. sphaericus. T h e 122 and 110 kD a proteins w ere d em o n strated n o t to be p art o f the p arasp o ral crystal and the 110 kD a protein not to be toxic to m osquito larvae (B ow dileh et a l, 1989; B au m an n and B aum ann, 1991; B aum ann et a l, 1991). T h erefo re, we are le ft w ith an o th er w ell characterized S -layer with no functional role y e t assigned. Even the putative protective role o f the S -layer against m uram idases w as not confirm ed (Stira et a l , 1990).
B a c t e r o i d e s b u c c a e , B a c te r o id e s s p p . , an d o t h e r p a t h o g e n s a s s o c ia te d w ith p e rio d o n titis
In th e p ro g ressio n o f th e in flam m ato ry p erio d o n tal d isease, th ere is a c le a r succession o f the resid en t m icrobiota from a predom inantly gram -p o sitiv e facultatively an aero b ic one, to one that is alm o st exclusively gram -negative and anaerobic, A m ajo r co m p o n e n t o f this co m m u n ity com prises several Bacteroides species frequently found in a sso ciatio n w ith apical periodontitis (infection o f the ja w b o n e). F ro m these, five sp e c ie s h av e been show n to possess S-layers: B. buccae, B, caplllus, B, pentosaceus
12
(H aap asalo et al,, 1985; K ornm an and H olt, 1981), B, heparinolyticus (O k u d a et al., 1985), and B, forsythus (T anner et at,, 1986). H ow ever, the S -lay er is n o t u su ally the o u term o st lay er on m ost o f these bacteria since they also po ssess slim e layers detected as an electro n o paque m aterial in specim ens stain ed in the p resen ce o f ta n n ic acid (H aap asalo et al., 1985) o r ruthenium red (K ornm an and H olt, 1981). M o reo v er, B. buccae and B, capillus also possess pill (H aapasalo etal,, 1985).
Sjhrgen et al, (1.985) show ed that two different types o f h exagonal S -layers are p resen t on d ifferen t strains o f B, buccae, B. capillus and B. pentosaceus. T h e lattice c o n sta n t o f o n e S-layer is 2'1.5 ± 0.5 nm and the o th er 7.7 ± 0.3 nm. A lthough the tw o S -la y c r ty p es can c o -e x ist on the sam e su p p o rtin g m em b ran e, a n aly sis by o p tical d iffraction sh o w ed that they did not o ccu r on top o f each o th er (su p erim p o sed layers). In th e sam e stu d y (SjOrgen etal,, 1985), the 3-D reco n stru ctio n o f th e 21.5 nm array w as achieved. It consisted o f a sin g le m orphological unit o f 6-fold sy m m e try w ith a cen tral pore o f ~5 nm in d iam eter and six sm aller pores (5 x 2.5 nm ) su rro u n d in g it. In ad d itio n to th ese tw o S -lay er types, cells o f B. buccae have been sh o w n to p o sses an h ex ag o n ally arranged periodic structure (K erosuo etal,, 1987 & 1988b). T h is kin d o f array , form ed by cry stallin e o u ter m em brane proteins, also has been o b serv ed in o th e r p a th o g e n ic b acteria like Bordetella pertussis (K essel et al,, 1988) and o th e r non- pathogcnic species (review ed in M essner and Sleytr, 1992). T h e relation betw een the 8
nm array o f crystalline ou ter m em brane proteins and the tw o S-layers is not know n. In the first rep o rt o f O kuda et al. (1985) no characterization o f the S -lay er o f B. heparinolyticus w as attem pted. Later, this w as structurally ch aracterized by K erosuo et al, (1 9 8 8 b ) as a thin electro n d en se lay er su rro u n d ed by a m o rp h o u s m aterial and separated from the outer m em brane by an electron dense, 20 nm gap. T h is layer has an hexagonal lattice w ith a spacing o f about 20 nm. T h e stru ctu re o f th e S -lay er fro m B. forsythus w as also fu rth er ch ara cterized by K erosuo (1 9 8 8 ) as an o b liq u e array o f
a b o u t 10 nm sp acin g and 10 nm height. S om etim es the array co u ld be sep arated in sin g le lines (ragged appearance), suggesting m ultiple su b u n it-su b u n it in teractio n s; the interactions w ithin lines being stronger than interactions betw een lines, U nfortunately, in th ese and oth er reports docum enting the existence o f S-laycrs on Bacteroides species ,no biochem ical characterization w as attem pted and only in one case, th a t o f B, buccae, w as the role o f the S-laycr in m ediating an interaction with leukocytes in vitro explored (K ero su o et al,, 1990). U nfortunately, no S -layer d eficient m utants w ere available and a sp ecific ro le for the S -laycr w as n o t dem onstrated. M o reo v er, so m e iso lates studied
13
w ere ap p a re n tly cap su lated (K, L ounatm aa, personal co m m u n icatio n ) o b scu rin g the role o f the S -lay er in adherence,
A n o th e r S -lay c r for w h ich a fu n ctio n al role has been ex p lo re d is th at o f Wollinella recta, an o ral pathogen frequently im plicated in m ixed periodontal infections to g eth er with o th e r flagellated, anaerobic gram -negatives; Campylobacter coacistts and Eikenella conodens (L ai et ah, 1981). From these organism s only W. recta clearly d isp la y ed an S -lay er o f hexagonal space sym m etry and a lattice co n stan t o f ab o u t 20 nm , D o k lan d et a l (1988 & 1990), as w ell as K ero su o et a l (1 9 8 8 a ) fu rth e r ch aracterized th e stru ctu re o f the S -layer from V/. recta, A close relationship betw een S -la y e r and o u te r m e m b ran e w a p fo u n d and a 3-D reco n stru c tio n w a s ach iev ed (D okland, 1988 & 1990), confirm ing the p6 sym m etry and a lattice co n stan t o f 21. nm , T h e th ick n ess o f the la y er w as 15 nm (u n u su ally thick) and it w as form ed by bilobed p ro te in su b u n its in an M6C3 arra n g e m en t (refer lo Fig, 1). T h e six h eav y dom ains a ro u n d the 6-fo ld sym m etry axis form a funnel-shaped m assive un it th at in teract w ith a d ja c e n t units th rough the lig h ter do m ain s at the 3-fold axis. In terestin g ly , a second p e rio d ic stru c tu re w as p resen t in n eg ativ ely stain ed cell w all frag m e n ts, Us 2-D p ro jectio n sh o w ed a p2 sym m etry w ith u n it cell vectors o f 8.5 x 12,5 nm and a relative angle o f 85°, an d apparently it represented an alternate crystallization lattice o f the sam e S -la y e r p ro tein (D o k lan d , 1988), C o m p arin g S -lay cr p o sitiv e and S -lay e r negative stra in s o f W, recta it w as sh o w n th at the p resen ce o f S -lay c r c o rrelate d w ith the ap p earan ce o f an acidic 142-154 kD a protein (B orinsky and H olt, 1990), T h e ab sence o f S -la y e r also cau sed a 45-60% in crease in bacterial ad h eren ce to a hum an gingival fib ro b last c e lt line. A lthough clearly pointing to a potentially im p o rtan t function, this alte re d b ac teria -h o st c e ll in teractio n in the ab sen ce o f S -lay er has n o t been fu rth er characterized. Interestingly, as previously indicated for Aeromonas species o f the 0 :1 1 s e ro ty p e , th e L P S o f W. recta p o s s e s s e s O -p o ly s a c c h a rid e c h a in s o f ra th e r h o m o g en eo u s size co n tain in g 5-8 rep eatin g units (G illespie e t a l , 1988). M oreover, this L P S had a n u nusual high co n ten t o f rham nose (88%).
T w o o th e r organism s associated w ith oral infections have been dem o n strated to p o sse ss S -layers. T h e se are Campylobacter sputorum and Bacillus sp, strain M 3 198, T h e fo rm e r c o n s titu te s a ca se in w h ich the p re se n c e o f S -la y e r h a s on ly been d o c u m en ted in thin sectio n s o f the o rg an ism , since its fine stru c tu re co u ld n o t be re so lv e d by n eg ativ e stain in g (L ai et a l, 1981). In terestin g ly (see b elo w ), the fin e stru c tu re o f th e S -lay er o f an o th er C am pylobacter, Campylobacter fetus, also has n o t
14
been resolved by n eg ativ e staining, T h e strain M 3198 is a p eritrich o u sly flag ellated , facu ltativ ely an aero b ic Bacillus species, related to li, coagulans and B, circulans. It w as isolated from a ro o t canal infection and found to have an oblique S -layer w ith 9.5 nm spacing betw een lines (H aapasalo etal., 1988).
B a c te r o id e s n o d o s u s
T his organism is the causal ag en t o f ovine fo o tro t disease (h o o f in fe ctio n s o f sh eep and g o ats). It is an o b lig ate an aero b ic, g ram -n eg ativ e rod w ith an u n certain tax o n o m ic g e n e ric p o sitio n , th a t has been p ro p o sed to be m o v e d o u t fro m th e Bacteroides g en u s (S h ah , 1992), to g e th e r w ith m an y o th e r b a c te ria p re v io u sly classified as Bacteroides species (actually, m ost o f the above m entioned oral path o g en s arc bein g reclassified into the genus Prevotella), T he cell surface o f this o rg an ism is very co m p lex , o w in g to the p resence o f pili, cap su le, u nusual ro d -lik e stru c tu res, a d iffu se m aterial associated w ith pili, polar rings and an S -layer (E v ery and S k erm an , 1980). T h e S -lay er is co m p o sed o f su b u n its arran g ed in a h ex ag o n al la ttice w ith a spacing o f 6-7 nm , clearly observed in negatively stained and freeze-etch ed sp ecim en s o f d iffe re n t v iru len t strains. A lthough the role o f surface structures in th e v iru len ce o f B, nodosus w as being investigated (E v ery and S k erm an , 1980), no fu rth er fin d in g s have been reported.
C a m p y l o b a c t e r f e t u s
T h e g e n u s Campylobacter en co m p asse s th irteen sp ecies o f g ra m -n e g a tiv e , sle n d e r c u rv e d ro d s, m o tile by m ean s o f a sin g le p o la r fla g ellu m . S p e c ie s o f Campylobacter are asacch aro ly tic and produce th eir en erg y th ro u g h resp iratio n and m etabolism o f am ino acids (T enovcr and F ennell, 1992). C fetus, the type sp ecies o f the genus, is an im p o rtan t veterinary pathogen cau sin g venereal genital tract in fectio n s in c a ttle th at m ay lead to infertility and abortion. In h u m an s in fe ctio n s are rare, b u t b eco m e sy ste m ic in c o m p ro m ised in d iv id u als. A lth o u g h m o st c a se s re p o rte d in hum ans involve extra intestinal infections, C, fetus also causes acute d iarrh eal illness,
T he first rep o rt on the existence o f an S -layer on C. fetus w as th at o f M cC o y et al, (1975). T h e S -layer w as ev id en t in thin sections, and in co n trast to oth er S -lay ers, a fu n ctio n al ro le as an an tip h ag o cy tic su rface stru ctu re w as read ily re c o g n ized . T w o ty p es o f lattice w ere ev id en t in the m icrographs o f negatively stain ed sp ecim en s la te r reported by M cC o y et al, (1976), but at that tim e this fact passed u n considered. It took
