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Use of the fluorescent probe 1-N-phenyl napthylamine to study the interactions of aminoglycoside antibiotics… Loh, Bernadette Anne 1984

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Use  o f the f l u o r e s c e n t probe  1-N-phenyl napthylamine  i n t e r a c t i o n s of aminoglycoside a n t i b i o t i c s  t o study the  w i t h the  o u t e r membrane o f Pseudomonas a e r u g i n o s a  by  B e r n a d e t t e Anne jLoh B.Sc,  U n i v e r s i t y o f B.C., 1980  A t h e s i s submitted i n p a r t i a l of  the requirements  fulfillment  f o r the degree o f  Master o f S c i e n c e r  in  The F a c u l t y o f Graduate  Studies  Department o f M i c r o b i o l o g y  We accept t h i s t h e s i s as conforming  t o the r e q u i r e d s t a n d a r d  The U n i v e r s i t y o f B r i t i s h  Columbia  September 1984 © Bernadette Anne Loh, 1984  »6  In  presenting  requirements of  British  it  freely  agree for  this for  available  that  I  by  understood  that  his  or  copying  gain  partial degree that  reference  for  purposes  or  financial  agree  for  permission  scholarly  in  an advanced  Columbia,  department for  thesis  fulfilment at  the  or  Library  copying  may b e g r a n t e d  by  s h a l l make  the  I of  s h a l l not  be  of  further this  thesis  head of  representatives. publication  the  University  and study.  extensive  her  the  of  It  this  allowed without  my  is thesis my  written  permission.  i  Department  of  MICROBIOLOGY  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  October  1 1 , 1984  Columbia  ABSTRACT  The mode o f i n t e r a c t i o n o f the p o l y c a t i o n i c  aminoglycoside  a n t i b i o t i c s w i t h the s u r f a c e o f Pseudomonas a e r u g i n o s a c e l l s was s t u d i e d u s i n g the hydrophobic  f l u o r e s c e n t probe 1-N-phenyl napthylamine  A d d i t i o n o f the a m i n o g l y c o s i d e of NPN l e d t o a s h i f t nm.  gentamicin to i n t a c t c e l l s  i n the presence  i n the f l u o r e s c e n c e e m i s s i o n maximum from 460 t o 420  A t the same time the NPN f l u o r e s c e n c e i n t e n s i t y  Gentamicin  (NPN).  increased four f o l d .  caused no such e f f e c t s when added t o o u t e r membrane v e s i c l e s  s u g g e s t i n g t h a t the i n c r e a s e d f l u o r e s c e n c e r e s u l t e d from the i n t e r a c t i o n of gentamicin with i n t a c t c e l l s .  Gentamicin-promoted NPN uptake 2+  i n h i b i t e d by the d i v a l e n t c a t i o n s Mg  2+ and Ca  , but o c c u r r e d i n the  absence o f g e n t a m i c i n t r a n s p o r t a c r o s s the i n n e r membrane. concentrations of gentamicin  The  independent  o f the g e n t a m i c i n  A c e n t r i f u g a t i o n t e c h n i q u e was used t o demonstrate  g e n t a m i c i n caused  a c t u a l uptake  r a t e o f NPN uptake sigmoidal fashion. aminoglycoside  At h i g h e r  (50 ug/ml) the i n c r e a s e o c c u r r e d w i t h i n a few seconds.  f i n a l f l u o r e s c e n c e i n t e n s i t y was almost  concentration.  Low  (2 ug/ml) caused NPN f l u o r e s c e n c e t o  i n c r e a s e over a p e r i o d o f 4 minutes i n a s i g m o i d a l f a s h i o n . concentrations  was  o f NPN from the supernatant.  that  The i n i t i a l  v a r i e d according t o the gentamicin concentration i n a S i m i l a r d a t a were o b t a i n e d f o r seven  antibiotics.  other  The d a t a when r e a n a l y s e d as a H i l l - p l o t  gave  a s e r i e s o f l i n e s w i t h a mean s l o p e ( t h e H i l l number) o f 2.26 ± 0.26, s u g g e s t i n g t h a t the i n t e r a c t i o n o f a m i n o g l y c o s i d e s with the c e l l s u r f a c e to p e r m e a b i l i z e i t t o NPN i n v o l v e d a t l e a s t 3 s i t e s and demonstrated  positive cooperativity.  There was  a statistically  significant  r e l a t i o n s h i p between the p s e u d o - a s s o c i a t i o n c o n s t a n t K p l o t s and  s  from the  Hill  the minimal i n h i b i t o r y c o n c e n t r a t i o n s f o r the 8 a n t i b i o t i c s  These r e s u l t s  are c o n s i s t e n t w i t h the concept  that  aminoglycosides  i n t e r a c t a t a d i v a l e n t c a t i o n b i n d i n g s i t e on the P. a e r u g i n o s a membrane and p e r m e a b i l i z e i t to the hydrophobic  probe  NPN.  outer  iv  Table  o f Contents Page  ABSTRACT  i i  TABLE OF CONTENTS List  . .  iv  of Figures  vi  L i s t of Tables  ix  ACKNOWLEDGEMENTS  X  INTRODUCTION  1  MATERIALS AND METHODS Bacterial  A  s t r a i n s and growth c o n d i t i o n s  Preparation of c e l l  suspension  4  A n t i b i o t i c s and chemicals Fluorescence  4  and p o l a r i z a t i o n measurements  . . . .  5  Measurement o f c e l l - b o u n d NPN  5  Preparation  6  o f o u t e r membrane v e s i c l e s and liposomes  RESULTS S e l e c t i o n o f a f l u o r e s c e n t probe  7  E f f e c t s o f growth media and b u f f e r s  27  M i c r o s t i r r e r Apparatus and A e r a t i o n E f f e c t  27  Cyanide pretreatment  28  of c e l l s  . . . .  G e n t a m i c i n enhancement o f NPN f l u o r e s c e n c e  and i n h i b i t i o n  by d i v a l e n t c a t i o n s  35  Uptake o f NPN  40  K i n e t i c s o f gentamicin-promoted NPN uptake Polarization  studies  . . .'  47 52  V  T a b l e o f Contents,  continued Page  DISCUSSION  57  LITERATURE CITED  62  vi  List  of Figures Page  1.  Effects  o f gentamicin  and magnesium on the f l u o r e s c e n c e  i n t e n s i t y o f ANS i n the presence o f P. a e r u g i n o s a c e l l s o r o u t e r membrane 2.  I n t r i n s i c emission membrane v e s i c l e s  3.  Excitation  Excitation  and emission  o u t e r membrane v e s i c l e s 5.  Fluorescence  8.  12  fluorescence  s p e c t r a o f ANS added t o  and the e f f e c t o f added  14  gentamicin.  a d d i t i o n on ANS f l u o r e s c e n c e  16  vesicles.  of phospholipid v e s i c l e s  E f f e c t of gentamicin membrane  i n the  vesicles.  pyrene, and the e f f e c t o f gentamicin 7.  10  H103  s p e c t r a o f ANS f l u o r e s c e n c e  E f f e c t o f magnesium and gentamicin in phospholipid  6.  s p e c t r a of outer  o f P. a e r u g i n o s a  presence o f o u t e r membrane 4.  intact  vesicles.  and e x c i t a t i o n  and e m i s s i o n  8  i n the presence o f  19  addition.  and magnesium on NPN f l u o r e s c e n c e  in  21  vesicles.  Time course  o f i n c r e a s e i n NPN f l u o r e s c e n c e  presence o f i n t a c t P. a e r u g i n o s a  cells  intensity  i n the  23  and d i f f e r e n t  2+ concentrations 9.  o f gentamicin  E f f e c t o f s e q u e n t i a l a d d i t i o n o f NPN on f l u o r e s c e n c e o f o u t e r membrane v e s i c l e s  10.  o r Mg  i n the presence o f  E f f e c t o f potassium s u c c i n a t e  (or glucose)  a e r a t i o n o f the c e l l suspension 11.  on NPN  gentamicin. a d d i t i o n and  29  fluorescence.  E f f e c t o f KCN treatment on the gentamicin i n NPN f l u o r e s c e n c e .  25  promoted r i s e  31  vii  L i s t of Figures,  continued Page  12.  (A)  Gentamicin-promoted i n c r e a s e i n NPN a d d i t i o n o f NPN  to i n t a c t c e l l s  presence o f sodium a z i d e .  fluorescence a f t e r  o f P. aeruginosa  (B)  33  i n the  I n h i b i t i o n by the  a d d i t i o n o f d i v a l e n t c a t i o n s o f the gentamicin-promoted i n c r e a s e i n NPN P. 13.  f l u o r e s c e n c e of  intact c e l l s of  aeruginosa.  E x c i t a t i o n and e m i s s i o n  s p e c t r a o f NPN f l u o r e s c e n c e i n  36  aqueous s o l u t i o n . 14.  Emission  and e x c i t a t i o n s p e c t r a o f NPN f l u o r e s c e n c e a f t e r  a d d i t i o n o f NPN the presence o f 15.  t o a suspension  o f P. a e r u g i n o s a  H103 i n  gentamicin.  I n h i b i t i o n o f the gentamicin-promoted enhancement o f NPN fluorescence  38  in intact cells  o f P. a e r u g i n o s a  41  H103 by  divalent cations. 16.  E f f e c t o f d i v a l e n t c a t i o n s on the i n i t i a l gentamicin-promoted i n c r e a s e i n NPN P. a e r u g i n o s a  17.  fluorescence of  K i n e t i c s o f gentamicin-promoted i n c r e a s e i n NPN  (MIC) o f the d i f f e r e n t 1 Q  aminoglycoside  of the p s e u d o a s s o c i a t i o n  from H i l l  47  cells.  R e l a t i o n s h i p between the minimal i n h i b i t o r y c o n c e n t r a t i o n s  log  43  cells.  f l u o r e s c e n c e of i n t a c t 18.  r a t e o f the  p l o t s such as those  a n t i b i o t i c s and the  constants  Ks, e x t r a p o l a t e d  shown i n F i g . 17.  50  vi i i  L i s t of F i g u r e s ,  19.  The  e f f e c t of gentamicin  intensity  of DPH  continued  a d d i t i o n on the p o l a r i z e d f l u o r e s c e n c e  added t o i n t a c t c e l l s of P.  aeruginosa.  54  ix  L i s t of Tables Page I.  Influence of gentamicin  c o n c e n t r a t i o n on the t o t a l uptake o f  NPN and enhancement of NPN  f l u o r e s c e n c e i n P.  45  aeruginosa  cells. II.  Fluorescence p o l a r i z a t i o n  o f the DPH taken up a f t e r the  interaction  and polymyxin B w i t h P.  cells.  of gentamicin  aeruginosa  55  ACKNOWLEDGEMENTS  I owe many thanks t o Dr. R.E.W. Hancock f o r h i s generous  advice,  support and encouragement throughout t h i s p r o j e c t . I would a l s o l i k e t o thank the members o f my s u p e r v i s o r y  committee,  Dr. D. S y e k l o c h a and Dr. R.A.J. Warren f o r t h e i r time and p a t i e n c e , and Susan Heming f o r h e r s k i l l f u l  t y p i n g o f t h i s manuscript.  xi  DEDICATION  This thesis me g r e a t support,  i s d e d i c a t e d w i t h g r a t i t u d e t o my parents who have g i v e n i n s p i r a t i o n and encouragement over the y e a r s .  1  INTRODUCTION  The  aminoglycosides  recognized  a r e a group o f p o l y c a t i o n i c a n t i b i o t i c s  i n the 1940s w i t h the d i s c o v e r y o f s t r e p t o m y c i n .  t h i s f a m i l y o f a n t i b i o t i c s e.g. gentamycin, proved v e r y e f f e c t i v e Aminoglycoside  i n the treatment  uptake  tobramycin,  o f many b a c t e r i a l  into b a c t e r i a l c e l l s  r e l a t i o n s h i p w i t h time and can be d i v i d e d  S i n c e then  kanamycin, has infections.  follows a sigmoidal  i n t o t h r e e phases ( 1 , 6 ) .  an i o n i c b i n d i n g i n t e r a c t i o n o c c u r s between the a m i n o g l y c o s i d e and the c e l l , and EDPII.  first  which i s f o l l o w e d by two energy  The i n i t i a l b i n d i n g a t the c e l l  First,  molecules  dependent phases c a l l e d EDPI  s u r f a c e i s r a p i d and  r e v e r s i b l e and tends t o n e u t r a l i z e the c e l l ' s n e t n e g a t i v e s u r f a c e charge (7).  The f i r s t  aminoglycosides  energy  dependent phase, EDPI, i n v o l v e s g r a d u a l uptake o f  (1,6,7).  EDPII begins when accumulation o f a m i n o g l y c o s i d e  becomes l i n e a r and r a p i d . I t has been suggested event  t o the c e l l s precedes  (7,10).  uptake  lethal  o r i s c o i n c i d e n t w i t h the onset o f EDPII  Both the EDPI and EDPII phases o f uptake  r e p r e s e n t uptake The  t h a t the a c t u a l  have been assumed t o  a c r o s s the e n e r g i z e d c y t o p l a s m i c membrane. of polycationic a n t i b i o t i c s ,  such as a m i n o g l y c o s i d e s ,  a c r o s s the o u t e r membrane o f Pseudomonas a e r u g i n o s a has been p o s t u l a t e d t o o c c u r v i a the s e l f promoted uptake  pathway (7,15).  T h i s uptake  i n v o l v e s the d i s p l a c e m e n t o f d i v a l e n t c a t i o n s e.g. M g the LPS by the p o l y c a t i o n i c a m i n o g l y c o s i d e s .  + +  or C a  + +  mechanism from  Thus, the d i v a l e n t c a t i o n s  c r o s s b r i d g i n g o f l i p o p o l y s a c c h a r i d e (LPS) i s d i s r u p t e d c a u s i n g d e s t a b i l i z a t i o n o r d i s t o r t i o n o f the membrane ( 7 ) .  localized  In support o f t h i s  2  h y p o t h e s i s the i n t e r a c t i o n o f s t r e p t o m y c i n or gentamicin w i t h P. a e r u g i n o s a causes lysozyme and  enhancement o f o u t e r membrane p e r m e a b i l i t y t o  i n c r e a s e d uptake  of the  fi-lactam,  nitrocefin  (8).  a d d i t i o n , e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA), which d i s r u p t s c r o s s b r i d g e s by c h e l a t i o n r a t h e r than displacement causes enhancement of uptake  o f lysozyme and n i t r o c e f i n  i n c r e a s e d r a t e of k i l l i n g  by a m i n o g l y c o s i d e s  Mg^  +  similar  (8) as w e l l as  (18).  In  Another  line  an of  evidence  i s t h a t o u t e r membrane-altered mutants o f P. a e r u g i n o s a , w i t h  apparent  decrease  to  EDTA, polymyxin  uptake  i n the number o f Mg-binding and a m i n o g l y c o s i d e s  pathway f o r a m i n o g l y c o s i d e s has  (14).  s i t e s show c r o s s - r e s i s t a n c e However, the s e l f - p r o m o t e d  not yet been demonstrated  Gram-negative b a c t e r i a and a m i n o g l y c o s i d e uptake may organisms  v i a the p o r i n p r o t e i n mediated  To o b t a i n f u r t h e r  i n f o r m a t i o n about  w i t h P. a e r u g i n o s a , we The  i n other  occur i n these  pathway (13). the i n t e r a c t i o n o f  decided to u t i l i z e  use o f f l u o r e s c e n t probes  aminoglycosides  f l u o r e s c e n t probes  as  tools.  t o study the s t r u c t u r e and f u n c t i o n o f  b i o l o g i c a l membranes i s w e l l documented (4,5,9,11).  Commonly used  i n biomembrane s t u d i e s are 1 , 8 - a n i l i n o - l - n a p t h a l e n e s u l p h o n i c a c i d and 1-N-phenyl naphthylamine  (NPN).  These probes  are p a r t i c u l a r l y  because they f l u o r e s c e weakly i n aqueous environments strongly fluorescent Furthermore,  i n n o n - p o l a r or hydrophobic  they are extremely  s o l v e n t , pH and temperature, given  assay.  an  probes (ANS) useful  but become v e r y  environments.  s e n s i t i v e to environmental  f a c t o r s such  and v e r y l i t t l e m a t e r i a l i s r e q u i r e d i n a  as  In t h i s t h e s i s , the r e s u l t s of a study u s i n g f l u o r e s c e n t probes t o l o o k a t the i n t e r a c t i o n o f a m i n o g l y c o s i d e s w i t h P. a e r u g i n o s a i s reported.  I have o b t a i n e d f u r t h e r e v i d e n c e t h a t  p e r m e a b i l i z e the c e l l interaction  aminoglycosides  a t the o u t e r membrane and that the s i t e  i s probably a d i v a l e n t c a t i o n binding s i t e .  of  4  MATERIALS AND METHODS  B a c t e r i a l s t r a i n s and growth s t r a i n H103  conditions.  Pseudomonas a e r u g i n o s a PA01  (14) was used i n a l l experiments.  p r o t e o s e peptone No. 2 medium ( D i f c o ) .  I t was grown i n 1% ( w t / v o l )  E x p e r i m e n t a l c u l t u r e s were  started  from an o v e r n i g h t b r o t h c u l t u r e and grown a t 37°C w i t h v i g o r o u s s h a k i n g to  an o p t i c a l d e n s i t y a t 600 nm (0D. .) o f 0.4 - 0.6. rt/  P r e p a r a t i o n of c e l l  suspension.  F i f t y ml of mid-log phase c e l l s were  c e n t r i f u g e d down a t 3000 x g f o r 10 min, and resuspended i n 5 mM Na Hepes b u f f e r pH 7.2, w i t h o r w i t h o u t 1 mM o suspension was l e f t  KCN, a t an 0D. of 0.5. 600 nn  This  cell  a t 23 C f o r 30-60 min b e f o r e adding any r e a g e n t s .  C o n t r o l experiments u s i n g P. a e r u g i n o s a s t r a i n H103 plasmid-encoded B-lactamase  (RPl) c o n t a i n i n g a  i n i t s p e r i p l a s m , demonstrated  t h a t d u r i n g the  time c o u r s e o f the experiments no B-lactamase l e a k e d out o f the c e l l , s u g g e s t i n g t h a t the o u t e r membrane remained A n t i b i o t i c s and c h e m i c a l s . kanamycin  Gentamicin s u l f a t e , neomycin  sulfate,  s u l f a t e and s t r e p t o m y c i n s u l f a t e were purchased from the Sigma  Chemical Co. ( S t . L o u i s , Mo.). r e c e i v e d from E l i L i l l y , a gift  intact.  Tobramycin  and n e t i l m y c i n s u l f a t e were  I n c . Canada (Scarborough, O n t a r i o ) .  Amikacin  was  from B r i s t o l - M y e r s Canada (Ottawa, O n t a r i o ) , w h i l e s i s o m y c i n  s u l f a t e was o b t a i n e d from the S c h e r i n g C o r p o r a t i o n ( P o i n t e Quebec).  NPN;  Claire,  1 , 6 - d i p h e n y l h e x a t r i e n e (DPH); and ANS were purchased  Sigma Chemical Co.  from  5  Fluorescence solution  and p o l a r i z a t i o n measurements.  ANS was made as a 60mM s t o c k  i n 0.9% (w/v) NaCl and used a t a f i n a l c o n c e n t r a t i o n o f 60 yM.  E x c i t a t i o n and e m i s s i o n r e s p e c t i v e l y with  slit  wavelengths f o r ANS were s e t a t 375 and 475 nm, widths o f 5nm.  NPN was d i s s o l v e d i n acetone a t a  c o n c e n t r a t i o n o f 500 yM and used a t a f i n a l c o n c e n t r a t i o n suspension) o f 10 yM.  C o n t r o l experiments showed no s i g n i f i c a n t  o f the added acetone on the r e s u l t s r e p o r t e d here. and  emission  fluorescence  spectrophotometer equipped with the c u v e t t e  slit  widths o f 5nm.  E x c i t a t i o n and  Fluorescence  i n t e n s i t i e s are g i v e n  arbitrary units.  a f t e r gentamicin  were prepared  The amount o f NPN bound t o c e l l s  before  treatment was determined by a m o d i f i c a t i o n o f the  c e n t r i f u g a t i o n technique ml)  650-10S  a Haake c i r c u l a t i n g water  h o l d i n g chamber a t 30°C.  Measurement o f c e l l - b o u n d NPN. and  spectra  wavelengths f o r NPN were u s u a l l y s e t a t 350 nm and 420nm  r e s p e c t i v e l y , with in  effect  Fluorescence  i n t e n s i t i e s were measured u s i n g a Perkin-Elmer  bath t o m a i n t a i n emission  (in c e l l  d e s c r i b e d by Nieva-Gomez e t a l . (16).  containing cyanide-treated  cells  Samples (4  (resuspended t o OD 600  = 0.5) and 10 yM NPN.  A l i q u o t s (1 ml) o f each sample were taken  and  a f t e r the a d d i t i o n o f g e n t a m i c i n  end  o f each experiment.  supernatant  and a t the  These a l i q u o t s were c e n t r i f u g e d a t 9,000 rpm f o r  1 min u s i n g an Eppendorf m i n i f u g e . c e l l s o r gentamicin  at varying concentrations,  before  C o n t r o l experiments without  were a l s o performed.  added  The NPN c o n c e n t r a t i o n o f the  was determined by measuring the f l u o r e s c e n c e  i n the presence  6  of  3% (v/v) T r i t o n X-100 and comparison  curves were l i n e a r over the range  with a standard curve.  o f NPN c o n c e n t r a t i o n s used  Standard  (0 - 10 uM).  P r e p a r a t i o n o f o u t e r membrane v e s i c l e s and l i p o s o m e s . Outer membranes from s t r a i n H103 were p r e p a r e d as p r e v i o u s l y d e s c r i b e d (14) and resuspended a t a f i n a l p r o t e i n c o n c e n t r a t i o n o f 5 mg/ml. s u s p e n s i o n was d i l u t e d  Twently  y l o f o u t e r membrane  i n 2 ml Hepes b u f f e r and s o n i c a t e d f o r 15-30 s e c .  2+ Mg  , g e n t a m i c i n and f l u o r e s c e n t probe were added as r e q u i r e d . V e s i c l e s were a l s o p r e p a r e d from c o m m e r c i a l l y o b t a i n e d l i p i d s  such as  p h o s p h a t i d y l c h o l i n e and p h o s p h a l i d y l e t h a n o l a m i n e and from H103 lipopolysaccharide (3)] .  (LPS) [prepared a c c o r d i n g t o method by Darveau, 1983  7  RESULTS  S e l e c t i o n o f a f l u o r e s c e n t probe. out  P r e l i m i n a r y experiments were c a r r i e d  w i t h 1,8-ANS, a probe commonly used i n membrane s t u d i e s .  (GM)  a d d i t i o n t o i n t a c t P. a e r u g i n o s a c e l l s  a substantial  i n the presence of ANS  caused  i n c r e a s e i n f l u o r e s c e n c e ; however, the k i n e t i c s o f t h i s  i n c r e a s e were complex and i n c l u d e d an i n i t i a l by a second, slower r a t e of i n c r e a s e These complex k i n e t i c s l e d me  o u t e r membrane v e s i c l e s had 350 and 430 nm  increase  followed  t o l o o k at a s i m p l e r system u s i n g  intrinsic  respectively  immediate  ( F i g . 1, curve A ) .  membrane or LPS v e s i c l e s and l i p o s o m e s .  of  Gentamicin  I t was  found t h a t both i n n e r and  e x c i t a t i o n and e m i s s i o n wavelengths  ( F i g . 2).  In the presence of  ANS,  e x c i t a t i o n was  shown as a broad band between 330-390 nm w h i l e the e m i s s i o n  wavelength was  r e d - s h i f t e d to 520 nm  ug/ml GM,  t h e r e was  an immediate  (Fig.  1, curve C ) , w i t h a s h i f t  (Fig.  4).  ( F i g . 3).  increase in fluorescence i n e m i s s i o n wavelength  V e s i c l e s p r e p a r e d from p h o s p h a t i d y l c h o l i n e ethanolamine  With the a d d i t i o n o f 50 intensity  back t o 470  nm  (PC) and p h o s p h a t i d y l  (PE) as w e l l as those p r e p a r e d from H 1 0 3 - l i p o p o l y s a c c h a r i d e  (LPS) showed a s i m i l a r response ( F i g . 5 ) . caused an immediate  enhancement of ANS  That i s , g e n t a m i c i n a d d i t i o n  f l u o r e s c e n c e and no  secondary  increase. 2+ Furthermore, d i v a l e n t c a t i o n s l i k e Mg i n c r e a s e i n ANS  2+ and Ca  f l u o r e s c e n c e ( F i g . 1, curve B; 5 ) .  caused a s i m i l a r  8  F i g u r e 1.  E f f e c t s of gentamicin  i n t e n s i t y o f ANS i n the presence membrane v e s i c l e s . and D) o r v e s i c l e s  and magnesium on the f l u o r e s c e n c e o f P. a e r u g i n o s a  i n t a c t c e l l s or outer  A t 0 min 5 yM ANS was added t o the c e l l s (B and C ) .  (curves A  S h o r t l y t h e r e a f t e r (as i n d i c a t e d by the  arrow) the f o l l o w i n g a d d i t i o n s were made:  Curve A - 20 yg/ml  gentamicin  added t o i n t a c t c e l l s ; Curve B - 10 yM MgCl^ added t o o u t e r membrane v e s i c l e s ; Curve C - 20 yg/ml g e n t a m i c i n  added t o o u t e r membrane  v e s i c l e s ; Curve D - no f u r t h e r a d d i t i o n s t o i n t a c t c e l l s to  (similar  results  curve D were o b t a i n e d when no a d d i t i o n was made t o o u t e r membrane  vesicles).  10  F i g u r e 2.  I n t r i n s i c e m i s s i o n and e x c i t a t i o n s p e c t r a o f o u t e r membrane  v e s i c l e s o f P. a e r u g i n o s a H103.  Outer membrane v e s i c l e s were  t o a f i n a l c o n c e n t r a t i o n o f 0.1 mg/ml p r o t e i n s o n i c a t e d f o r 30 s e c .  f o r e m i s s i o n and e x c i t a t i o n scans.  In the  t h e e m i s s i o n and e x c i t a t i o n maxima were 430 nm (A)  and 350 nm (B) r e s p e c t i v e l y . membrane v e s i c l e s .  i n 2 ml o f 5 mM Hepes and  E x c i t a t i o n and e m i s s i o n wavelengths were s e t a t 370  nm and 478 nm r e s p e c t i v e l y absence o f added probe,  resuspended  S i m i l a r v a l u e s were o b t a i n e d w i t h  inner  11  370 nm  478 nm  12  F i g u r e 3.  E x c i t a t i o n and e m i s s i o n s p e c t r a  p r e s e n c e o f o u t e r membrane V e s i c l e s .  o f ANS  f l u o r e s c e n c e i n the  Outer membrane v e s i c l e s were  resuspended t o a f i n a l c o n c e n t r a t i o n o f 100 yg/ml i n 2 ml o f 5 mM Hepes.  ANS was added t o 60 yM and e x c i t a t i o n  were s e t a t 375 and 480 nm r e s p e c t i v e l y .  E m i s s i o n and e x c i t a t i o n maxima  were 520 nm (A) and a broad band o f 330-390 nm scans were o b t a i n e d w i t h  inner  membranes.  and e m i s s i o n wavelengths  (B) r e s p e c t i v e l y .  Similar  13  480 nm 1  375 nm * 520 nm  A  330 nm  B  14  F i g u r e 4.  Excitation  (A) and  emission  added to o u t e r membrane v e s i c l e s Excitation  60 yM 470  and  gentamicin  385  nm  Hepes.  to 50 yg/ml.  respectively.  ANS  was  Emission  ANS  gentamicin.  and 475  Outer membranes were resuspended to a f i n a l  ug/ml i n 2 ml of 5 mM and  the e f f e c t of added  and e m i s s i o n wavelengths were s e t at 375  respectively. o f 100  and  (B) f l u o r e s c e n c e s p e c t r a of  nm concentration  added to a c o n c e n t r a t i o n of and  e x c i t a t i o n maxima were  Inner membrane v e s i c l e s gave s i m i l a r  results.  375nm  740 nm i  475 nm  385 nm  16  F i g u r e 5.  E f f e c t o f magnesium and g e n t a m i c i n  in phospholipid vesicles.  Phosphatidylcholine  resuspended t o 100 yg/ml i n 2 ml of 5 mM 30 s e c .  ANS was added a t 60 yM.  added t o 0.1 mM values and  and gentamicin  of fluorescence  LPS v e s i c l e s .  a d d i t i o n on ANS vesicles  fluorescence  (PCy) were  Hepes b u f f e r and s o n i c a t e d f o r  Subsequently M g  2 +  (MgCl > was 2  to 20 yg/ml f i n a l c o n c e n t r a t i o n .  i n t e n s i t y were seen w i t h  Similar  phosphatidylethanolamine  17  0  2  A  6  8  TIME (min)  10  12  U  18  Presumably, the e f f e c t s o f g e n t a m i c i n i n t a c t c e l l s were c o m p l i c a t e d negatively-charged uptake o f ANS.  enhancement o f f l u o r e s c e n c e i n  by charge n e u t r a l i z a t i o n o f  ANS by p o l y c a t i o n i c aminoglycosides  allowing further  Such e f f e c t s were p r e v i o u s l y demonstrated f o r d i - a n d  trivalent cations  (5,11).  A second f l u o r e s c e n t probe, pyrene, was found t o be r a p i d l y taken up by p h o s p h o l i p i d v e s i c l e s but t h i s f l u o r e s c e n c e was not s t a b l e and almost instantaneously probably  s t a r t e d t o decay ( F i g . 6 ) .  had a v e r y  short fluorescence  t h e r e f o r e not s u i t a b l e .  T h i s suggested t h a t pyrene  lifetime  Subsequent gentamicin  i n t h i s system and was a d d i t i o n d i d not enhance  fluorescence. In c o n t r a s t t o these  data,  the f l u o r e s c e n c e o f the n e u t r a l probe NPN,  was enhanced upon the i n t e r a c t i o n o f i n t a c t c e l l s w i t h  gentamicin  curve A) and a f t e r p l a t e a u i n g remained s t a b l e f o r 10-20 min. although  ( F i g . 8,  However,  NPN i t s e l f was r a p i d l y taken up by o u t e r membrane and  phospholipid v e s i c l e s , very l i t t l e  or no f l u o r e s c e n c e enhancement was 2+  observed upon a d d i t i o n o f g e n t a m i c i n ,  Mg  2+ o r Ca  (Fig. 7).  This  suggested t h a t NPN was s p e c i f i c a l l y r e p o r t i n g on an i n t e r a c t i o n o f gentamicin  with  the s u r f a c e o f i n t a c t c e l l s .  Therefore,  NPN was chosen  f o r a l l subsequent s t u d i e s . In a d d i t i o n , the s e q u e n t i a l a d d i t i o n o f NPN t o o u t e r membrane v e s i c l e s caused an i n c r e a s e i n f l u o r e s c e n c e  from 1-5 ym NPN, a peak o r  p l a t e a u a t 7-8 ym NPN, and s l i g h t lower f l u o r e s c e n c e e m i s s i o n NPN c o n c e n t r a t i o n s  t o 10 ym ( F i g . 9 ) .  at higher  19  F i g u r e 6. pyrene, vesicles  Fluorescence of p h o s p h o l i p i d v e s i c l e s  and the e f f e c t o f g e n t a m i c i n a d d i t i o n . (PC^) were resuspended  b u f f e r and s o n i c a t e d f o r 30 s e c .  Pyrene was  added a t 2.5  added a t 20 yg/ml.  vesicles  results.  of  Phosphatidylcholine  t o 100 ug/ml i n 2 ml o f 5 mM  s u b s e q u e n t l y g e n t a m i c i n was (PE ) gave s i m i l a r  i n the presence  um  Hepes and  Phosphatidylethanolamine  20  '  0  1  1  — i  2  1  6 TIME (min) k  1  8  r -  10  21  F i g u r e 7.  E f f e c t o f g e n t a m i c i n o r magnesium a d d i t i o n on NPN f l u o r e s c e n c e  i n membrane v e s i c l e s . of  Inner o r o u t e r membranes were resuspended  5 mM Hepes b u f f e r i n t h e presence o f 10 yM NPN.  t o 1 mM and g e n t a m i c i n t o 20 yg/ml as r e q u i r e d .  i n 2 ml  Magnesium was added  22  8  IM 1  NPN  IM  GM i  1  NPN •  TIME  Mg  GM i  OM  i  NPN 1  Mg GM  23  F i g u r e 8. presence  Time course o f i n c r e a s e i n NPN  fluorescence intensity  i n the  o f i n t a c t P. a e r u g i n o s a c e l l s and d i f f e r e n t c o n c e n t r a t i o n s o f  g e n t a m i c i n or Mg^ . +  were made:  At the arrow l a b e l l e d GM the f o l l o w i n g a d d i t i o n s  Curve A - 20 yg/ml o f g e n t a m i c i n ; Curve B - 2 yg/ml o f  g e n t a m i c i n ; Curve C - 2 yg/ml o f g e n t a m i c i n D - no g e n t a m i c i n  and 100 yM M g C l ; Curve 2  added ( r e s u l t s were i d e n t i c a l whether or not MgCl^ was  added i n the absence o f g e n t a m i c i n ) .  C e l l s were p r e t r e a t e d w i t h 1 mM  as d e s c r i b e d i n the below (see F i g . 11).  KCN  t-O  25  F i g u r e 9.  E f f e c t o f s e q u e n t i a l a d d i t i o n o f NPN on f l u o r e s c e n c e o f o u t e r  membrane v e s i c l e s resuspended  i n the presence o f g e n t a m i c i n .  i n 2 ml o f 5 mM Hepes b u f f e r  at 20 yg/ml (arrow B ) .  Outer membranes were  (arrow A ) .  Gentamicin was added  NPN was then added s e q u e n t i a l l y ,  yM (arrow C) and c o n t i n u i n g from 1-10 yM.  s t a r t i n g a t 0.5  Fluorescence  to  27  E f f e c t s of d i f f e r e n t overnight  growth media and  c u l t u r e of s t r a i n H103  l e v e l of i n t r i n s i c  a d d i t i o n of NPN  20 um  C e l l s grown and w i t h low Mg^ ) presence of NPN. fluorescence BM2  No  broth  Resuspension of  increase  unstable  i n NB-grown c e l l s ,  but  with  (minimal media  fluorescence  i n the NPN  a decrease i n f l u o r e s c e n c e  of  grown c e l l s . Of the d i f f e r e n t media used f o r growing and  growth i n PP2  both o v e r n i g h t  c e n t r i f u g a t i o n proved to be consistency. and  observed  G e n t a m i c i n a d d i t i o n r e s u l t e d i n a moderate  increase  of  ug/ml.  resuspended i n n u t r i e n t b r o t h or BM2 h i g h but  high  to the p r o d u c t i o n  i n f l u o r e s c e n c e was  at 20  an  r e s u l t e d i n a very  (presumably due  or g e n t a m i c i n  showed a f a i r l y  +  i n PP2  fluorescence  endogeneous f l u o r o p h o r e s ) .  buffers.  allowed  and  resuspension  resuspending  i n 5 mM  cells,  Hepes buffer? a f t e r  the b e s t c o m b i n a t i o n f o r convenience  and  C o n t r o l "Experiments gave comparable r e s u l t s from day repeated  results  M i c r o s t i r r e r Apparatus and spectrophotometer was  to be  to  day  analyzed.  Aeration Effect.  equipped w i t h  The  fluorescence  a m i c r o s t i r r e r apparatus  (Lawrence  Instruments, Ltd.) which c o n s i s t e d of a t i n y magnetic s t i r r e r , u n i t attached  under the c u v e t t e  continuous  s t i r r i n g of the  holder.  o b j e c t i v e was  to f i n d out i f  sample would ensure more e f f e c t i v e  between probe m o l e c u l e s and r e s u l t s among d i f f e r e n t  Our  bacterial cells  experiments.  fluctuations  in fluorescence  fluorescence  i n any  level  particular  and  sample.  and  contact  produce more c o n s i s t e n t  U n e x p e c t e d l y , t h i s caused a generally higher  prolonged  baseline  Many f a c t o r s c o u l d be  responsible  28  for this  i n c l u d i n g a e r a t i o n of the  magnetic f l e a bar  i n the  sample and  light  s c a t t e r from  cuvette.  In a d d i t i o n , manual a g i t a t i o n / a e r a t i o n (shaking) suspension  after reaching  a carbon source  ( F i g . 10). E. c o l i  Again,  these  cell caused  same e f f e c t was  phenomena were r e m i n i s c e n t  the  observed  (1 mM)  was  added  of s i m i l a r s t u d i e s i n  (2) .  of c e l l s .  r e s u l t s were not c o m p l i c a t e d cytoplasmic  to ensure t h a t the  shown i n F i g . 11,  the  EDPII) g e n t a m i c i n initial  1 mM  demonstrated a c o n t i n u i n g f l u o r e s c e n c e whereas i n n o n - t r e a t e d  cells,  fluorescence  (1 mM) the  initial  (1,5).  the  confirmed  However, c y a n i d e - t r e a t e d  i n c r e a s e was  state  f o l l o w e d by  Another energy  the d e c l i n e i n f l u o r e s c e n c e  by cyanide  cells was a  without  S i m i l a r phenomena ( i . e . a f l u o r e s c e n c e  which would be b l o c k e d  under  inhibitor,  d e c r e a s e ) have been observed i n E. c o l i .  s e c r e t i o n of NPN,  As  was  r a t e s of t h e gentamicin-promoted i n c r e a s e  ( F i g . 12A).  f o l l o w e d by a steady energized  also prevented  T h i s was  i n c r e a s e u n t i l a steady  i n f l u o r e s c e n c e to b a s e l i n e l e v e l s .  influencing  the  which  r a t e of i n c r e a s e of f l u o r e s c e n c e  i n the presence or absence of c y a n i d e .  sodium a z i d e  on  potassium c y a n i d e  t r a n s p o r t and k i l l i n g  a v a r i e t y of the c o n d i t i o n s t e s t e d below.  achieved,  observed  by e i t h e r the e f f e c t s of g e n t a m i c i n  pretreated c e l l s with  (both EDPI and  identical  In o r d e r  membrane d u r i n g t r a n s p o r t or by post-uptake e f f e c t s on. c e l l  m e t a b o l i s m ( 7 ) , we  decline  The  such as p o t a s s i u m s u c c i n a t e or g l u c o s e  Cyanide pretreatment  blocks  of the  the maximum f l u o r e s c e n c e e m i s s i o n  f l u o r e s c e n c e to d e c r e a s e to i t s base l e v e l . if  the  and  in  NPN  increase an  or a z i d e i n  29  F i g u r e 10.  E f f e c t of potassium succinate  a e r a t i o n o f the c e l l labelled  suspension  10 yM NPN.  D-glucose) was added t o 2 mM. to aerate  on NPN f l u o r e s c e n c e .  1, i n t a c t c e l l s o f P. a e r u g i n o s a  5 mM Hepes b u f f e r w i t h  the c e l l  suspensions.  again u n t i l i t achieved  ( o r glucose)  a d d i t i o n and A t the arrow  H103 were resuspended i n 2 ml o f  A t arrow 2, potassium s u c c i n a t e ( o r  A t arrow 3, the c u v e t t e was shaken manually A t l a t e r times the f l u o r e s c e n c e  a pre-aeration l e v e l of fluorescence.  rose  31  F i g u r e 11.  E f f e c t of KCN  fluorescence. cells.  treatment on the g e n t a m i c i n promoted r i s e  At 0 min 5 yM NPN was  added t o i n t a c t P.  At the arrow 20 yg/ml g e n t a m i c i n was  resuspended i n 1 mM addition.  KCN  added.  i n NPN  aeruginosa  Curve A - c e l l s  i n Hepes b u f f e r and h e l d f o r 10 min p r i o r t o NPN  Curve B - c e l l s  resuspended i n Hepes b u f f e r .  33  F i g u r e 12.  (A)  a d d i t i o n o f NPN azide. mM  Gentamicin-promoted i n c r e a s e i n NPN to i n t a c t c e l l s  g e n t a m i c i n was (B)  c e l l s were resuspended i n 2 ml o f 5  and 1 mM  Na a z i d e .  At arrow 2,  added to 10 yg/ml.  I n h i b i t i o n by the a d d i t i o n o f d i v a l e n t c a t i o n s o f the g e n t a m i c i n -  promoted i n c r e a s e i n NPN the  after  of P. a e r u g i n o s a i n the presence o f sodium  At the arrow l a b e l l e d 1, H103  Hepes b u f f e r w i t h 10 yM NPN  fluorescence  fluorescence of i n t a c t c e l l s  arrow l a b e l l e d 1, H103  P. a e r u g i n o s a .  i n t a c t c e l l s were resuspended i n 2 ml o f 5 mM 2+  Hepes b u f f e r w i t h 10 yM NPN 2+ Mg  and 1 mM  Na a z i d e .  At arrow 2, Mn  ,  2+ o r Ba  At  were added t o 5 mM.  A t arrow 3, g e n t a m i c i n was  a f i n a l c o n c e n t r a t i o n of 10 yg/ml.  added t o  35  t h i s case, was p o s t u l a t e d t o be r e s p o n s i b l e f o r these subsequent experiments were performed wth cyanide k i n e t i c s and i d e n t i c a l  initial  effects  (2). A l l  due t o the s i m p l e r  r a t e s i n the presence o r absence o f  cyanide.  Gentamicin enhancement o f NPN f l u o r e s c e n c e cations.  and i n h i b i t i o n by d i v a l e n t  C o n t r o l experiments were performed t o demonstrate t h a t 10 uM  NPN was the i d e a l n o n - l i m i t i n g c o n c e n t r a t i o n e f f e c t s of gentamicin  on c e l l s .  c y a n i d e - t r e a t e d P. a e r u g i n o s a increase i n fluorescence ( F i g . 8, curve 340  D).  The a d d i t i o n o f 10 yM NPN t o  s t r a i n H103 c e l l s caused an immediate  small  i n t e n s i t y above the background l e v e l o f the c e l l s  At t h i s  nm and the e m i s s i o n  f o r v i s u a l i z a t i o n o f the  stage the e x c i t a t i o n wavelength maximum was  wavelength maximum was 460 nm, s i m i l a r t o the  maxima observed w i t h NPN added t o aqueous s o l u t i o n ( F i g . 1 3 ) . When gentamicin  was added, the e m i s s i o n  fluorescence process.  i n t e n s i t y a t t h i s wavelength i n c r e a s e d i n a time dependent  The e x c i t a t i o n wavelength maximum s h i f t e d t o 350 nm ( F i g . 1 4 ) .  When c e l l s 350  i n the presence o f 10 yM NPN were e x c i t e d by l i g h t o f  nm wavelength and the e m i s s i o n  kinetics of fluorescence gentamicin  maximum s h i f t e d t o 420 nm and the  added.  a t 420 nm f o l l o w e d over time, the  increase v a r i e d with  A t low gentamicin  the c o n c e n t r a t i o n o f  concentrations  (2 yg/ml-below t h e  minimal i n h i b i t o r y c o n c e n t r a t i o n f o r s t r a i n H103 c e l l s grown under  these  c o n d i t i o n s ) , the enhancement o f NPN f l u o r e s c e n c e was b i p h a s i c ( F i g . 8, curve  B ) . At h i g h e r  concentrations  o f gentamicin eg. 20 yg/ml, the  i n c r e a s e o f NPN f l u o r e s c e n c e was r a p i d and p l a t e a u e d w i t h i n 20 sec a f t e r a  36  F i g u r e 13. solution.  Excitation  and e m i s s i o n s p e c t r a  A s o l u t i o n o f 5 yM NPN  o f NPN f l u o r e s c e n c e i n aqueous  i n 5 mM Hepes was made.  E m i s s i o n and  e x c i t a t i o n maxima were o b s e r v e d a t 460 (A) and 340 nm (B) r e s p e c t i v e l y .  38  F i g u r e 14. addition  E m i s s i o n and e x c i t a t i o n s p e c t r a o f NPN f l u o r e s c e n c e a f t e r  o f NPN t o a suspension o f P. a e r u g i n o s a H103 i n the presence o f  gentamicin.  H103 c e l l s were resuspended i n 5 mM Hepes b u f f e r w i t h 10 yM  NPN and 20 yg/ml g e n t a m i c i n . observed  E m i s s i o n and e x c i t a t i o n maxima were  a t 420 nm (A) and 350 nm (B) r e s p e c t i v e l y .  40  4 fold  increase i n fluorescence emission  fluorescence  The  i n c r e a s e i n the presence o f 100 ug/ml gentamicin  instantaneous  was  almost  and f l u o r e s c e n c e d i d not decay over time ( d a t a not shown).  The c o n c e n t r a t i o n o f g e n t a m i c i n fluorescence  ( F i g . 8, curve A ) .  added i n f l u e n c e d only the k i n e t i c s of  i n c r e a s e and not the steady  s t a t e l e v e l achieved  (see F i g . 8,  curves A and B ) . The a d d i t i o n o f the d i v a l e n t c a t i o n s , Mg and  Sr  2+  at low l e v e l s  (eg. 50 yM Mg  2+  , Ca  corresponding  , Ba  2+  , Mn  and Na a z i d e - p r e t r e a t e d  o f 0 - 50 yM Mg  d e c r e a s e s i n the i n i t i a l  2+  C; F i g . 15)  o f NPN f l u o r e s c e n c e .  2+ Increasing concentrations  2+  , F i g . 8, curve  i n h i b i t e d the enhancement, by g e n t a m i c i n , e f f e c t was observed i n both c y a n i d e  2+  This  cells.  2+ o r Ca  produced  r a t e o f NPN  fluorescence  enhancement ( F i g . 16A).  I n c r e a s i n g the gentamicin c o n c e n t r a t i o n i n the 2+ 2+ presence of a f i x e d amount o f Ca o r Mg l e d to a gradual increase in  the i n i t i a l 2+  Ca  r a t e of f l u o r e s c e n c e  ( F i g . 16B).  As mentioned above,  2+ and Mg  o r any o f the o t h e r  c a t i o n s used, d i d not themselves  cause enhancement o f NPN f l u o r e s c e n c e  i n whole c e l l s or p h o s p h o l i p i d  vesicles. Uptake o f NPN.  The i n c r e a s e o f NPN f l u o r e s c e n c e upon gentamicin  c o u l d have two e x p l a n a t i o n s ;  i t c o u l d r e p r e s e n t NPN being  the c e l l s from the supernatant  addition  taken up  o r i t c o u l d be due to an a l t e r a t i o n  environment of the NPN r e s u l t i n g  i n f l u o r e s c e n c e enhancement.  ( T a b l e I) suggested t h a t a combination o f these r e s p o n s i b l e f o r the observed f l u o r e s c e n c e  two e f f e c t s  increase.  into i n the  Our data  was  The amount of NPN  41  Figure  15.  I n h i b i t i o n o f the gentamicin-promoted enhancement  fluorescence At  in intact cells  o f P. a e r u g i n o s a H103  the arrow l a b e l l e d 1, H103  o f NPN  .  by d i v a l e n t c a t i o n s .  c e l l s were resuspended i n 5 mM  Hepes b u f f e r  2+ w i t h 1 mM  KCN.  At arrow 2, Mg  was  added to 5 mM.  added t o 10 yM and a t arrow 4, g e n t a m i c i n was  At arrow 3, NPN  was  added t o a f i n a l  c o n c e n t r a t i o n o f 20 yg/ml. S i m i l a r r e s u l t s were o b t a i n e d w i t h 2+ 2+ 2+ 2+ o f Mn , Ca , Ba or Sr t o 5 mM.  addition  Fluorescence o  •-NO  m  oo  43  F i g u r e 16.  E f f e c t o f d i v a l e n t c a t i o n s on the i n i t i a l  r a t e o f the  gentamicin-promoted i n c r e a s e i n NPN f l u o r e s c e n c e o f P. a e r u g i n o s a  cells.  ' 2+ 2+ In p a n e l A i n c r e a s i n g c o n c e n t r a t i o n s o f Mg ( A ) and Ca (•) were added t o s e p a r a t e Methods.  cuvettes containing c e l l s  Immediately afterwards  and NPN as d e s c r i b e d i n  20 yg/ml g e n t a m i c i n  was added t o each  c u v e t t e and the k i n e t i c s o f f l u o r e s c e n c e i n c r e a s e f o l l o w e d over p a n e l B e i t h e r 50 yM Mg  2+  ( A ) , 40 yM Ca  2+  time.  (•) o r no c a t i o n (0)  were added p r i o r to the a d d i t i o n of v a r y i n g amounts o f g e n t a m i c i n f i n a l c o n c e n t r a t i o n g i v e n on the X a x i s . on c y a n i d e - t r e a t e d  cells.  In  t o the  A l l experiments were performed  44  45  Table  I.  Influence KPN  of gentamicin  c o n c e n t r a t i o n on the t o t a l uptake o f  and enhancement o f NPN  fluorescence  C e l l bound NPN i n  Gentamicin  Arbitrary  Concentration (ug/ml)  (umol NPN  Total  Units taken  i n P. a e r u g i n o s a  fluorescence  of c e l l up)  a  cells.  bound  Calculated  NPN  in A r b i t r a r y units'  fluoresence enhancement o f  5  cell  a  Cell  2  10.3 ± 0.6  (1.7)  17.7 ± 4.6  1.7  5  12.4 ± 4.5  (2.0)  23.3 ± 4.4  1.9  10  12.0 ± 0.8  (1.9)  26.3 ± 2.4  2.2  20  14.0 + 1.8  (2.1)  23.4 ± 2.4  1.7  50  17.7 + 4.6  (2.5)  29.7 ± 2.0  1.7  bound NPN was  gentamicin and  bound NPN  (after  c a l c u l a t e d by d e t e r m i n i n g  the f r e e NPN  the a d d i t i o n of g e n t a m i c i n .  of the NPN remaining  The r e s u l t s  u n i t s were c o n v e r t e d  constructed  by a d d i t i o n o f 3% T r i t o n X-100 t o d i f f e r e n t  sample a f t e r g e n t a m i c i n [Prior  to c e l l s  on  (column 2 ) .  This  to a standard  and i s the f l u o r e s c e n c e  i n t e n s i t y o f the c e l l  intensity prior  to  gentamicin  a d d i t i o n , 4 f l u o r e s c e n t u n i t s (0.3 umol o f NPN)  fluorescence  i s the i n c r e a s e  above the e x p e c t e d f l u o r e s c e n c e  curve  amounts o f NPN.  average].  C a l c u l a t e d as the r a t i o of t o t a l  after  i n f l u o r e s c e n c e measured i n the 10 min  a d d i t i o n minus the f l u o r e s c e n c e  to g e n t a m i c i n  cells  10 min  deviations) in arbitrary  t o umol NPN taken up by r e f e r e n c e  ^ T o t a l f l u o r e s c e n c e was the a c t u a l i n c r e a s e a f t e r a d d i t i o n of g e n t a m i c i n  i n the s u p e r n a t a n t  (means + s t a n d a r d  fluorescent  associated with  i n the absence o f  removal o f c e l l s by c e n t r i f u g a t i o n and a d d i t i o n o f 3% T r i t o n X-100)  s u b t r a c t i n g the d e t e r m i n a t i o n  addition.  c  (column 3) to c e l l  i n the f l u o r e s c e n c e o f c e l l  i n 3% T r i t o n X-100.  bound  NPN  bound NPN over and  were  46  bound to c e l l s b e f o r e  and  c e n t r i f u g a t i o n technique  a f t e r gentamicin  treatment was  u s i n g T r i t o n X-100.  determined by  T r i t o n X-100  was  previously  demonstrated to enhance o n l y the f l u o r e s c e n c e of c e l l - f r e e NPN e f f e c t on c e l l bound NPN approximately  (16).  4 f l u o r e s c e n t u n i t s of NPN  A f t e r a d d i t i o n of gentamicin reach  a steady  state l e v e l ,  f o u r to seven f o l d .  The  and  The  total  were a s s o c i a t e d w i t h  sufficient  concentration  cell  bound NPN  i n c r e a s e must have been due  to the NPN  i n c o r p o r a t e d i n t o an environment i n which i t was than i n T r i t o n X-100  uptake - thus the t o t a l f l u o r e s c e n c e  only occurred  was  this  uptake).  increased  The  lifetime  concentration,  suggesting  that  p a r t i t i o n e d i n t o a s i m i l a r environment i n a l l experiments.  u s i n g gentamicin to  3 of Table I ) .  i n c r e a s e i n f l u o r e s c e n t y i e l d of the c e l l bound  K i n e t i c s of gentamicin-promoted NPN  evaluate  how  concentrations the r a t e of NPN  uptake.  A s e r i e s of experiments,  between 1 and uptake was  be  as a consequence of  i n c r e a s e r e f l e c t s NPN  almost independent of the gentamicin  the NPN  fold.  being  I; N.B.  i n c r e a s e i n f l u o r e s c e n c e might have been caused by an The  f o l d range of  more h i g h l y f l u o r e s c e n t  s o l u t i o n s (see column 4, Table  f l u o r e s c e n c e enhancement a p p a r e n t l y  of the probe ( 9 ) .  relatively  a d d i t i o n c o u l d not  (compare columns 2 and  to  increased  bound f l u o r e s c e n c e changed o n l y 1.7  accounted f o r by the c e l l bound NPN Thus, p a r t of the  was  s i n c e over a 25  i n c r e a s e i n f l u o r e s c e n c e a f t e r gentamicin  no  cells.  time to a l l o w f l u o r e s c e n c e  maximum l e v e l of c e l l  concentrations,  but had  gentamicin,  c e l l - a s s o c i a t e d f l u o r e s c e n c e had  independent of the gentamicin gentamicin  In the absence of added  a  20 yg/ml, were performed  a f f e c t e d by the  gentamicin  NPN  47  F i g u r e 17. of  K i n e t i c s of gentamicin  intact c e l l s .  expressed weight),  i n a r b i t r a r y f l u o r e s c e n c e u n i t s per min taken  i n yg/ml.  cells.  fluorescence  In p a n e l A the i n i t i a l r a t e of f l u o r e s c e n c e i n c r e a s e per mg  cell  p l o t t e d a g a i n s t the c o n c e n t r a t i o n of  A l l experiments were performed w i t h  T h i s d a t a was  which the g e n t a m i c i n  dry  reanalyzed  a c c o r d i n g to a H i l l  c o n c e n t r a t i o n was  converted  gentamicin  cyanide-treated plot  to molar  ( p a n e l B) i n concentrations  and <(> i s the r a t i o of the r a t e of f l u o r e s c e n c e i n c r e a s e at the gentamicin  c o n c e n t r a t i o n t o the maximal r a t e of f l u o r e s c e n c e  given  increase  e x t r a p o l a t e d from p a n e l A.  The  Hill  plot) i s given  The  Y a x i s i n t e r c e p t i s equal to - l n  The  i n p a n e l B.  correlation coefficient  t h i s and  (V  from a s e r i e s of experiments l i k e those d e p i c t e d i n curves  A and B of F i g . 8, was (GM)  promoted i n c r e a s e i n NPN  a l l other H i l l  number (n = the s l o p e of the  Hill K. g  f o r the l i n e a r r e g r e s s i o n of the d a t a f o r  p l o t s was  g r e a t e r than  0.99.  49  concentration present.  We o b t a i n e d a f a m i l y o f s i g m o i d a l curves o f (eg. F i g . 8,  f l u o r e s c e n c e i n c r e a s e over time a f t e r g e n t a m i c i n  addition  curves A and B ) .  interactions,  attempted  o n l y t o analyze the i n i t i a l  increase. produced  Due t o the c o m p l e x i t y  A p l o t o f these i n i t i a l a sigmoidal plot  o f these  we  r a t e s o f the NPN f l u o r e s c e n c e  r a t e s a g a i n s t gentamicin c o n c e n t r a t i o n  ( F i g . 17A).  From t h i s ,  i t appeared  that a  c o o p e r a t i v e i n t e r a c t i o n had o c c u r r e d between gentamicin and c e l l s r i s e t o the uptake o f NPN,  giving  i e . t h e i n t e r a c t i o n o f one molecule o f  g e n t a m i c i n w i t h the c e l l s u r f a c e enhanced subsequent i n t e r a c t i o n s . d a t a were r e p l o t t e d as a H i l l distinguish the H i l l  plot  ( F i g . 17B) which a l l o w s one t o  simple, m u l t i p l e o r c o o p e r a t i v e i n t e r a c t i o n s .  plot  i s r e f e r r e d t o as the H i l l  number.  The s l o p e o f  T h i s number  i s usually  i n t e r p r e t e d as the approximate minimum number o f b i n d i n g s i t e s . gentamicin,  the H i l l  The  number was 1.95 ± 0.3 (average o f 5  For  experiments)  i n d i c a t i n g a c o o p e r a t i v e i n t e r a c t i o n w i t h a minimum o f 2 - 3 i n t e r a c t i o n sites.  Similar linear H i l l  d i f f e r e n t aminoglycoside d e r i v e d from H i l l  the 30 a n a l y s e s was  the H i l l  antibiotics.  calculated.  g  i n t e r a c t i o n c o e f f i c i e n t , one can c a l c u l a t e  Ks.  from  v a l u e ( p s e u d o - a s s o c i a t i o n c o n s t a n t ) f o r the  i n t e r a c t i o n o f gentamicin w i t h c e l l s , -ln  numbers  A mean and s t a n d a r d d e v i a t i o n o f 2.26 ± 0.26 f o r  addition to t h i s plot a K  I n t e r e s t i n g l y , the H i l l  p l o t analyses o f these d a t a were v e r y s i m i l a r among the  eight aminoglycosides.  In  p l o t s were o b t a i n e d w i t h each o f e i g h t  The c a l c u l a t e d K  s i n c e the Y - a x i s  intercept i s  v a l u e s were used f o r r e l a t i v e comparisons o f s the d i f f e r e n t a m i n o g l y c o s i d e s s i n c e they v a r i e d over 4 o r d e r s o f  50  F i g u r e 18. (MIC)  R e l a t i o n s h i p between the minimal  o f the d i f f e r e n t  aminoglycoside a n t i b i o t i c s  pseudo-association constants K , g  those shown i n F i g . 17. antibiotics (0);  and t h e i r  The  and the 1°8^Q o f the  e x t r a p o l a t e d from H i l l p l o t s such as  a b b r e v i a t i o n s f o r the  symbols were:  ( A ) ; neomycin = NM  aminoglycoside  tobramycin = TM  s i s o m y c i n = SI ( x ) ; g e n t a m i c i n = GM  netil-ymycin = NT  i n h i b i t o r y concentrations  ( A ) ; amikacin = AK  (•); s t r e p t o m y c i n = SM  ( f ) ; kanamycin = KM  (!) .  (•);  6H  KM SM oNM o o  4l  o o  AK 2H  • •  • •  SI *  •  • • •  A•  •  •  T  X  NT  • •  GM  TB  —i— 1  —i—  10 MIC (um)  100  52  magnitude.  A p l o t of l o g K  i n h i b i t o r y concentrations i n F i g u r e 18.  g  a g a i n s t the l o g a r i t h m o f the minimal  (MIC) f o r the d i f f e r e n t aminoglycosides  i s shown  There were s i g n i f i c a n t v a r i a t i o n s f o r each a n t i b i o t i c i n  i n d i v i d u a l determinations  o f the K  d e s p i t e the f a c t t h a t these  numbers  s d e r i v e d from H i l l  p l o t s i n which the c o r r e l a t i o n c o e f f i c i e n t s  ( r ) were  g r e a t e r than 0.99 and, as noted above, the s l o p e s o f the i n d i v i d u a l (the H i l l  number) were remarkably c o n s t a n t .  might be d i f f e r e n t i a l c o n t a m i n a t i o n  One p o s s i b l e reason  lines  for this  o f i n d i v i d u a l batches o f c e l l s by  d i v a l e n t c a t i o n s s i n c e the i n i t i a l r a t e of NPN uptake was  strongly  a f f e c t e d by d i v a l e n t c a t i o n s ( F i g . 8, curve C; F i g . 15).  Nevertheless,  h i g h l y s i g n i f i c a n t c o r r e l a t i o n between l o g MIC and l o g K  was  a  s demonstrated ( r = 0.68, d f = 29, p <0.001 f o r a s t r a i g h t l i n e w i t h of 1.33 by l e a s t  squares a n a l y s i s ) .  interaction site  i n a cooperative  the SQ  the X a x i s i n t e r c e p t o f the H i l l  The a f f i n i t y of a s u b s t r a t e f o r i t s  interaction  or substrate concentration  a slope  i s sometimes expressed  a t h a l f maximal v e l o c i t y  plot = log S  demonstrate a s i g n i f i c a n t c o r r e l a t i o n between  Q  ^.  as  ( g i v e n by  We were a l s o a b l e t o and MIC f o r d i f f e r e n t  aminoglycosides. P o l a r i z a t i o n Studies. by gentamicin those and  A d d i t i o n o f the n e u t r a l probe DPH t o c e l l s  a d d i t i o n caused s i m i l a r k i n e t i c s of f l u o r e s c e n c e  shown i n F i g . 8 f o r NPN  ( F i g . 19).  followed  i n c r e a s e as  The enhancement o f NPN  (Table I )  DPH f l u o r e s c e n c e a f t e r gentamicin-promoted uptake suggested t h a t  probes were p a r t i t i o n i n g  i n t o a more f l u i d environment.  To c o n f i r m  we performed f l u o r e s c e n c e p o l a r i z a t i o n s t u d i e s with DPH, which has  these this,  53  f r e q u e n t l y been used f o r such s t u d i e s . polarization  i n acetone gave a low  v a l u e ( T a b l e I I ) s i n c e the probe i s h i g h l y m o b i l e i n t h i s  solvent  (Freifelder,  results  in increased polarization  T a b l e I I f o r DPH antibiotic.  DPH  1982).  Decreasing mobility  o r b i n d i n g o f the probe  ( F r e i f e l d e r , 1982)  i n water and c e l l - a s s o c i a t e d  DPH  as demonstrated i n  i n the absence o f added  In the presence of g e n t a m i c i n , or another  a n t i b i o t i c polymyxin 8, a decrease i n p o l a r i z a t i o n suggested t h a t t h e s e a n t i b i o t i c s environment w i t h i n the  cells.  caused DPH  was  polycationic observed.  t o move i n t o  This  a more mobile  54  F i g u r e 19.  The  fluorescence S t r a i n H103 yM a -  on the  added to i n t a c t c e l l s  c e l l s were resuspended i n 2 ml of 5 mM  fluorescence  i n t e n s i t y with  polarizer  fluorescence output  c -  i n t e n s i t y of DPH  addition  polarized of P.  aeruginosa.  Hepes b u f f e r w i t h  2  DPH.  output b -  e f f e c t of gentamicin  input p o l a r i z e r  s e t at 90° and  the  0°.  i n t e n s i t y with  polarizer  fluorescence  s e t at  the  s e t at 90  o  th input p o l a r i z e r  s e t at 90° and  the  .  i n t e n s i t y with  the  input p o l a r i z e r  s e t at 0° and  the  the  input p o l a r i z e r  s e t at 0° and  the  o output d -  polarizer  fluorescence output  s e t at 90  .  i n t e n s i t y with  polarizer  s e t at 0 ° .  At the arrow, 25 yg/ml g e n t a m i c i n for  d and  the f l u o r e s c e n c e  was  added w i t h  the p o l a r i z e r s  i n c r e a s e f o l l o w e d over 12  min.  s e t as  Fluorescence  Ln Ln  Table I I :  Fluorescence p o l a r i z a t i o n  o f the DPH taken up  a f t e r the i n t e r a c t i o n o f g e n t a m i c i n and polymyxin B w i t h P. a e r u g i n o s a  Experimental Conditions  cells.  Polarization  DPH  i n water  0.16  DPH  i n acetone  0.05  DPH c e l l s DPH + c e l l s + g e n t a m i c i n  0.36 (25 yg/ml)  DPH + c e l l s + polymyxin B (20 yg/ml)  0.27 0.28  57  DISCUSSION  In t h i s t h e s i s , the e f f e c t i v e n e s s o f f l u o r e s c e n t probes the i n t e r a c t i o n s o f AG's w i t h P. a e r u g i n o s a c e l l s has been S i n c e experiments  w i t h the a n i o n i c probe ANS produced  kinetics,  such t h a t the probe d i d not s p e c i f i c a l l y  happening  at the o u t e r s u r f a c e o f P. a e r u g i n o s a c e l l s ,  experiments  were c a r r i e d out w i t h NPN.  The uptake  i n studying demonstrated.  r a t h e r complex  i n d i c a t e what was a l l subsequent  o f NPN t h a t was  measured p r o b a b l y r e f l e c t s o u t e r membrane p e r m e a b i l i z a t i o n to NPN by amino glycosides.  As evidence f o r t h i s , enhancement o f NPN uptake  c o u l d o n l y be demonstrated  in intact bacterial c e l l s .  g e n t a m i c i n d i d not s t i m u l a t e uptake vesicles,  We found  that  o f NPN i n t o b a c t e r i a l o u t e r membrane  i n n e r membrane v e s i c l e s o r p h o s p h o l i p i d liposomes  synthetically.  by g e n t a m i c i n  prepared  In o t h e r words, the i n c r e a s e i n NPN f l u o r e s c e n c e upon  a d d i t i o n o f GM was independent v e s i c l e s became immediately  of gentamicin concentration.  These  s a t u r a t e d w i t h NPN, which had s p e c t r a l  p r o p e r t i e s s i m i l a r t o the NPN taken up by g e n t a m i c i n - t r e a t e d whole T h i s suggests t h a t g e n t a m i c i n way t h a t NPN can p a r t i t i o n membrane.  i s d i s o r g a n i z i n g the c e l l  cells.  s u r f a c e i n such a  i n t o the o u t e r (and p r o b a b l y a l s o the i n n e r )  Presumably the o u t e r membrane s t r u c t u r e was d i s o r g a n i z e d by the  French Press treatment  used t o make o u t e r membrane v e s i c l e s , and s y n t h e t i c  p h o s p h o l i p i d v e s i c l e s are simple l i p i d  bilayer  liposomes.  In f u r t h e r agreement w i t h t h i s concept, t h e r a t e o f NPN f l u o r e s c e n c e i n c r e a s e was r e l a t e d t o the amount o f g e n t a m i c i n added ( F i g s . 8, 1 7 ) , although the f i n a l  amount o f NPN a s s o c i a t e d w i t h c e l l s was r e l a t i v e l y  58  constant.  T h i s suggests t h a t NPN e n t e r s  number o f access until  areas,  the b a c t e r i a l c e l l v i a a l i m i t e d  but t h a t NPN uptake, once i n i t i a t e d ,  the s i t e s w i t h which NPN a s s o c i a t e s are s a t u r a t e d .  number o f access gentamicin  sites  proceeds  Presumably the  f o r NPN i s determined by the c o n c e n t r a t i o n o f  i n the system.  I t was p r e v i o u s l y proposed i n t h i s l a b , t h a t the i n t e r a c t i o n s i t e s f o r aminoglycosides  on the o u t e r membrane o f P. a e r u g i n o s a  where c a t i o n s n o n c o v a l e n t l y  c r o s s b r i d g e adjacent  Presumably, aminoglycosides  d i s p l a c e Mg^  d i s t o r t i n g o u t e r membrane s t r u c t u r e . 2+ demonstrate t h a t Mg NPN ( F i g . 8,16).  +  are those  LPS molecules  from these  sites  I n agreement w i t h  sites  (14,15).  thus  t h i s , we c o u l d  2+ o r Ca  i n h i b i t e d gentamycin-promoted uptake o f  T h i s r e s u l t c o u l d be e x p l a i n e d by c o m p e t i t i o n  p o l y c a t i o n i c a n t i b i o t i c gentamicin  o f the  and the d i v a l e n t c a t i o n s f o r a d i v a l e n t  c a t i o n b i n d i n g s i t e on the o u t e r membrane. Evidence has been p u b l i s h e d which suggests t h a t both the p o l y c a t i o n i c a n t i b i o t i c polymyxin B and the d i v a l e n t c a t i o n c h e l a t o r EDTA i n t e r a c t the same d i v a l e n t c a t i o n b i n d i n g s i t e as aminoglycosides In f u r t h e r agreement w i t h  like  with  gentamicin.  t h i s both polymyxin B (12) and EDTA (9)  cause an i n c r e a s e i n the f l u o r e s c e n t i n t e n s i t y o f hydrophobic f l u o r e s c e n t probes added t o c e l l s .  Helgerson  and Cramer (9) p o s t u l a t e d t h a t EDTA  treatment removed the p e r m e a b i l i t y b a r r i e r o f the o u t e r membrane o f E. c o l i  t o NPN.  T h i s would a l l o w the NPN m o l e c u l e s g r e a t e r access t o  b i n d i n g s i t e s on the c e l l polymyxin B i n t e r a c t s w i t h  surface.  More r e c e n t l y , i t has been shown t h a t  a d i v a l e n t c a t i o n b i n d i n g s i t e on LPS ( 1 7 ) .  S c h i n d l e r and Osborn (17) used f l u o r e s c e n c e  analysis of dansylated  d e r i v a t i v e s o f LPS t o show t h a t LPS c o n t a i n s one and p o s s i b l y two types o f binding s i t e s f o r M g  + +  and C a  + +  ,  i n the KDO and phosphate groups.  Furthermore, i t has been shown t h a t polymyxin B and gentamicin  each  compete w i t h a c a t i o n i c s p i n l a b e l probe, CAT.^, f o r a d i v a l e n t c a t i o n b i n d i n g s i t e on P. a e r u g i n o s a manuscript  LPS (AA P a t e r s o n ,  in preparation).  One o f the major o b s e r v a t i o n s of  o f t h i s paper i s t h a t the i n i t i a l  f l u o r e s c e n c e i n c r e a s e v a r y a c c o r d i n g t o the gentamicin  a f a s h i o n t h a t i s amenable t o k i n e t i c of  REW Hancock, E J McGroarty,  analysis.  sigmoidal p l o t suggesting  positive cooperativity.  r e p l o t t e d the d a t a as a H i l l t h a t the o r d i n a t e term  plot.  rates  c o n c e n t r a t i o n s gave a  To c o n f i r m t h i s ,  we  The advantage o f t h i s treatment i s  [ l o g (l-o))/o), where <J> i s the r a t i o o f the  rate of fluorescence increase at a given gentamicin u n i t s and thus  concentration i n  A p l o t of i n i t i a l  f l u o r e s c e n c e i n c r e a s e as a f u n c t i o n o f gentamicin  rates  c o n c e n t r a t i o n ] , has no  i s independent o f the a r b i t r a r y f l u o r e s c e n t u n i t s .  Thus,  assuming t h a t the r a t e o f NPN uptake d i r e c t l y r e f l e c t s the i n t e r a c t i o n o f gentamicin  with o u t e r membranes, which seems l i k e l y , the H i l l  p r o v i d e s k i n e t i c d a t a which r e f l e c t o n l y t h i s arguments are v a l i d  f o r each o f the e i g h t aminoglycosides  k i n e t i c a n a l y s i s by t h i s method. aminiglycosides  interaction.  In H i l l  i n c o r p o r a t i n g 30 s e p a r a t e  (the slope o f the H i l l  plot  Similar subjected to  p l o t analyses, of a l l eight s e t s o f d a t a , the H i l l  p l o t ) was 2.26 ± 0.26.  Since t h i s  number  p r o v i d e s an e s t i m a t i o n o f the minimum number o f i n t e r a c t i o n s i t e s in  number  involved  the c o o p e r a t i v e p e r m a e a b i l i z a t i o n o f o u t e r membranes t o NPN, we can  make the assumption t h a t a t l e a s t  t h r e e o r more s i t e s are i n v o l v e d .  While  the H i l l  numbers were remarkably  a m i n o g l y c o s i d e s , i t was varied substantially.  interesting The h i g h l y  similar  f o r each o f the 8  that the p s e u d o a s s o c i a t ion c o n s t a n t Ks  significant  r e l a t i o n s h i p between the Ks v a l u e and the MIC  (p < 0.001) l i n e a r v a l u e f o r the  different  aminoglycosides suggests t h a t the measured i n t e r a c t i o n at the s u r f a c e of the outer membrane may  be an important determinant  and/or  In agreement w i t h t h i s , M g  r a t e of k i l l i n g .  s t r o n g l y antagonize the t r a n s p o r t of and k i l l i n g also s i g n i f i c a n t l y  ( F i g . 8, curve C, 16).  our experiments  were performed  energy-dependent  is  intriguing  and C a ^  + +  +  which  uptake  (1)  by  I t s h o u l d be noted, however, t h a t  i n the presence of c y a n i d e which b l o c k s  t r a n s p o r t and k i l l i n g ,  outer membrane precedes  efficiency  by a m i n o g l y c o s i d e s  i n h i b i t e d the enhancement o f NPN  aminoglycosides  of the  these energy  suggesting that  dependent e v e n t s .  t h a t the time course o f NPN  uptake  interaction  at the  In t h i s r e g a r d , i t  following  the  treatment  of c e l l s w i t h a m i n o g l y c o s i d e s s t r o n g l y mimicked the time c o u r s e of aminoglycoside uptake Our experiments  (1,7).  a l s o suggested t h a t the i n c r e a s e i n NPN f l u o r e s c e n c e  a f t e r gentamicin a d d i t i o n to c e l l s was taken up i n t o the c e l l s environment Furthermore,  o f NPN  and  due  to a combination  ( s u b s e q u e n t l y ) an a l t e r a t i o n  m o l e c u l e s which enhanced i t s f l u o r e s c e n c e y i e l d .  polarization  caused the probe m o l e c u l e s  value.  being  i n the  s t u d i e s w i t h DPH,  another n e u t r a l f l u o r e s c e n c e  probe, confirmed t h a t the presence of g e n t a m i c i n i n a c e l l  environment  o f NPN  to p a r t i t i o n  w i t h i n the c e l l s ,  Alternatively  i n t o a more h i g h l y m o b i l e o r  as shown by a decrease  t h i s decrease  suspension fluid  in polarization  i n p o l a r i z a t i o n might  be e x p l a i n e d by  61  an a l t e r a t i o n on our  i n f l u o r e s c e n c e l i f e t i m e although we  c o u l d not measure t h i s  apparatus.  Although  t h i s does not f o r m a l l y demonstrate  that  i n t e r a c t w i t h o u t e r membranes t o promote t h e i r own  aminoglycosides  uptake, we  have  d e s c r i b e d the enhancement a f t e r g e n t a m i c i n treatment of the uptake hydrophobic B-lactam  probes  NPN  antibiotic  s e l f - p r o m o t e d uptake  and DPH,  nitrocefin hypothesis  hypothesis,  i t will  the i n i t i a l  interaction.  antibiotics directly  of  and p r e v i o u s l y o f the p r o t e i n lysozyme (8).  T h i s d a t a i s thus c o n s i s t e n t w i t h  (7,15).  To f u r t h e r t e s t and prove  be n e c e s s a r y t o l e a r n more about Attempts  the events  t o f l u o r e s c e n t l y tag the  are c u r r e n t l y underway f o r t h i s  reason.  and the  this  following  aminoglycoside  62  LITERATURE CITED  1. Bryan, L.E. and H.M. van den E l z e n . i n s u s c e p t i b l e and r e s i s t a n t Pseudomonas a e r u g i n o s a .  1976. S t r e p t o m y c i n  s t r a i n s o f E s c h e r i c h i a c o l i and  A n t i m i c r o b . Agents and Chemother.  2. Cramer, W.A., P.W. Postma. and S.L. Helgerson. of  N-phenyl-l-napthylamine  Escherichia c o l i .  accumulation  1976. An e v a l u a t i o n  as a probe o f membrane energy  Biochim.  9:928-938.  state i n  Biophys. A c t a 449:401-411.  3. Darveau, R.P. , Hancock, R.E.W.  1983. Procedure  f o rIsolation of  B a c t e r i a l L i p o p o l y s a c c h a r i d e s from both Smooth and Rough a e r u g i n o s a and S a l m o n e l l a typhimurium s t r a i n s .  Pseudomonas  J. Bacteriol.  155:831-838. 4. F r e i f e l d e r , D. and Co.  1982. " P h y s i c a l B i o c h e m i s t r y " , 2nd ed.  San F r a n c i s c o ,  562p.  5. Gomperts, B., F. Lantelme, and R. Stock. r e a c t i o n s w i t h b i o l o g i c a l membranes, 1,8-ANS.  J . Membr. B i o l .  6. Hancock, R.E.W.  1970. Ion a s s o c i a t i o n  s t u d i e d w i t h t h e f l u o r e s c e n t Dye  3:241-246.  1981. Aminoglycoside  uptake and mode o f a c t i o n -  w i t h s p e c i a l r e f e r e n c e t o s t r e p t o m y c i n and g e n t a m i c i n . and mutants.  W.H. Freeman  I. Antagonists  A n t i m i c r o b . Chemother. 8:249-276.  7. Hancock, R.E.W.  1981. Aminoglycoside  uptake and mode o f a c t i o n -  w i t h s p e c i a l r e f e r e n c e t o s t r e p t o m y c i n and g e n t a m i c i n . I I . E f f e c t s o f aminoglycosides  n cells.  A n t i m i c r o b . Chemother. 8:429-445.  63  8. Hancock, R.E.W., V . J . R a f f l e , and T . I . N i c a s . the o u t e r membrane i n g e n t a m i c i n in Pseudomonas 9. Helgerson, cell  and s t r e p t o m y c i n  uptake and k i l l i n g  A n t i m i c r o b . Agents Chemother. 19:7 7 7-783.  S.L., and Cramer, W.A.  envelope  caused  aeruginosa.  1981. Involvement o f  1977. Changes i n E s c h e r i c h i a c o l i  s t r u c t u r e and the s i t e s o f f l u o r e s c e n c e probe b i n d i n g  by c a r b o n y l cyanide  p-trifluoromethoxypahneylhydrazone.  Biochem. 16:4109-4117. 10. Hurwitz,  C , and C.L. Rosano.  permease. 11. Madeira, of  J. Bacteriol.  +  +  f o r a streptomycin  90:1233-1237.  V.M.C., and M.C. Antunes-Madeira.  C a ^ and Mg^ w i t h  Acta  1965. Evidence  1973. I n t e r a c t i o n  s y n a p t i c plasma membranes. Biochim.  Biophys.  323:396-407.  12. Newton, B.A.  1954. S i t e o f a c t i o n o f polymyxin on Pseudomonas  a e r u g i n o s a : Antagonism by c a t i o n s . 13. Nakae, R., and T. Nakae. antibiotics  J . Gen. M i c r o b i o l .  10:491-499.  1982. D i f f u s i o n o f a m i n o g l y c o s i d e  a c r o s s the o u t e r membrane o f E s c h e r i c h i a c o l i •  A n t i m i c r o b . Agents Chemother. 22:554-559. 14. N i c a s , T . I . , and R.E.W. Hancock. Pseudomonas  aeruginosa:  1980. Outer membrane p r o t e i n HI o f  Involvement i n a d a p t i v e and m u t a t i o n a l  r e s i s t a n c e t o e t h y l e n e d i a m i n e t e t r a a c e t a t e , polymyxin B, and gentamicin.  J. Bacteriol.  143:872-878.  15. N i c a s , T . I . and R.E.W. Hancock. to  1983. A l t e r a t i o n o f s u s c e p t i b i l i t y  EDTA, polymyxin B and g e n t a m i c i n  i n Pseudomonas  a e r u g i n o s a by  d i v a l e n t c a t i o n r e g u l a t i o n o f o u t e r membrane p r o t e i n HI. Microbiol.  129:509-517.  J . Gen.  64  16.  Nieva-Gomez, D. , J . Konisky, and R.B. Gennis. in E s c h e r i c h i a c o l i energy t r a n s d u c t i o n .  17.  Schindler, cations  induced by c o l i c i n  1976.  Membrane changes  I a and agents known t o d i s r u p t  Biochem. 15:2747-2753.  M., and M.J. Osborn.  1979.  I n t e r a c t i o n of d i v a l e n t  and polymyxin B w i t h l i p o p o l y s a c c h a r i d e .  Biochem.  18:4425-4430. 18. Sykes, R., and A. M o r r i s . to a n t i m i c r o b i a l drugs. 19.  U r a t a n i , V.  1982.  1975.  R e s i s t a n c e o f Pseudomonas  Progr. Med. Chem. 333:393.  Dansyl c h l o r i d e l a b e l l i n g o f Pseudomonas  a e r u g i n o s a t r e a t e d with P y o c i n RI: Change i n p e r m e a b i l i t y envelope.  aeruginosa  J. Bacteriol.  149:523-528.  o f the c e l l  

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