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Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas Aeruginosa Sawyer, Janet Gail 1987

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INTERACTION OF MACROPHAGE CATIONIC PROTEINS WITH THE OUTER MEMBRANE OF PSEUDOMONAS AERUGINOSA  By  JANET GAIL SAWYER  B.Sc,  University  o f Guelph, 1 9 8 5  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY  We a c c e p t t h i s t h e s i s as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA July 1987 ©  Janet G a i l Sawyer, 1 9 8 7  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or by his or her representatives.  It is understood that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  M^BOS'6UJ£M  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  OU.L4 2 i  ABSTRACT Purified functional membrane  proteins  and lipopolysaocharide of  were  derivative  of  Pseudomonas  polymyxin B  to bind to saturation to p u r i f i e d  similar H215  oationlo  used  ln  assays to determine t h e i r i n t e r a c t i o n s with the outer  fluorescent found  macrophage  affinity  A  (dansyl-polymyxin)  was  lipopolysaocharide,with  f o r the aminoglycoside  and wild type s t r a i n H103  aeruginosa.  supersensitive  lipopolysaocharide.  strain  MCP-1  could  displace more dansyl-polymyxln bound to the lipopolysaocharide of both s t r a i n s , whole  oells  polymyxin. examined  were  used,  Effects by  hydrophobic was  and bound with greater a f f i n i t y than MCP-2.  on  6.5.  displaced  bound  dansyl-  the  i n i t i a l rate  fluorescent probe  of  uptake  1-N-phenylnaphthylamine.  were  of  the  Uptake  enhanced i n the presence of MCPs, i n d i c a t i n g permeabilization  40 ug/ml,  ug/ml. with  also  the outer membrane of whole o e l l s  determining  of the outer membrane. at  MCPs  When  MCP-1  caused maximal uptake of the probe  MCP-2 at 70 ug/ml,  Uptake of the probe was  maximal  and crude extraot at  only  found to be enhanced at a d d  uptake occurring with only 7.5 jog/ml MCP-1  at  20 pH, pH  The data suggested that MCPs aot to permeablllze the outer  membranes of P_j_  aeruginosa  i n a manner analagous to that defined  for other polycationic agents.  ii  TABLE OF CONTENTS  Page.  ABSTRACT  i i  TABLE OF CONTENTS L i s t of Figures L i s t of Tables  i i i iv v  ACKNOWLEDGEMENTS  vi  INTRODUCTION  1  MATERIALS AND METHODS Macrophage/granulocyte procurement 5 Peptide p u r i f i c a t i o n 6 B a c t e r i a l s t r a i n s and growth conditions 7 A n t i b a c t e r i a l assay. 7 LPS i s o l a t i o n 8 Dansyl-polymyxin binding experiments 8 Binding i n h i b i t i o n experiments 9 Permeabilization of whole c e l l s to 1-N-phenylnaphthylamin e 10 Enhancement of phagocytosis by MCPs 10 RESULTS P u r i f i c a t i o n of MCPs A n t i b a c t e r i a l assay Dansyl-polymyxin binding t o p u r i f i e d LPS I n h i b i t i o n o f dansyl-polymyxin binding t o LPS by polycations Dansyl-polymyxin binding and i n h i b i t i o n to whole c e l l s Enhancement of 1-N-phenylnaphthylamine uptake by MCPs Enhancement of phagocytosis  12 13 18 27 32 32 46  DISCUSSION  49  LITERATURE CITED  54  iii  LIST OF FIGURES Page 1.  P-10 column f r a c t i o n a t i o n  14  2.  MCP p u r i f i c a t i o n by reversed phase FPLC  16  3.  LPS-dependent fluorescence of dansylpolymyxin  21  4.  H i l l plot of dansyl-polymyxin binding t o LPS..23  5. 6.  I n h i b i t i o n o f dansyl-polymyxin binding t o LPS by MCPs 28 Binding of dansyl-polymyxin t o i n t a c t cells...33  7.  I n h i b i t i o n of dansyl-polymyxin binding t o whole c e l l s by MCPs  37  8.  Enhancement of NPN uptake by i n t a c t c e l l s  39  9.  MCP-1 promoted enhancement of NPN f l u o r escence i n i n t a c t c e l l s at varying pH  44  iv  LIST OF TABLES Page 1. 2. 3. 4. 5. 6. 7.  Examples of b a c t e r i c i d a l c a t i o n i o proteins of phagocytes  2  K i l l i n g of P. aeruginosa s t r a i n s by crude macrophage extract and p u r i f i e d peptides  19  K i n e t i c s of binding of dansyl-polymyxin to P. aeruginosa LPS  25  I n h i b i t i o n by various polycations of dansylpolymyxin binding to P_j_ aeruginosa LPS  30  I n h i b i t i o n by various polycations of dansylpolymyxin binding to whole c e l l s  35  H i l l numbers and S 's f o r interactions of MCPs with P^ aeruginosa  42  Q  Phagocytosis of untreated and MCP-coincubated P. aeruginosa by u n e l i c i t e d rabbit alveolar macrophages 47  v  ACKNOWLEDGEMENTS  I  wish to thank Dr.  understanding thanks  and  Joe Lam and Dr.  encouragement  during  Bob Hancock f o r t h e i r my  studies.  Special  also to Nancy Martin and Niamh K e l l y f o r t h e i r  constant  help, support and friendship.  I  g r a t e f u l l y acknowledge the support of a Medical  Council Studentship  1986-87.  vi  Research  INTRODUCTION  Although  phagocytosis by macrophages constitutes the  mechanism  by  aspirated  bacteria  biochemical  which  non-specific i s achieved  mechanisms  resistance  (8),  to  major  infection  our understanding  of  of b a c t e r i a l k i l l i n g i s s t i l l  far  by the  from  complete (29). Mechanisms of macrophage antimicrobial k i l l i n g f a l l into two distinct  categories,  independent. the  namely,  oxygen-dependent  Oxygen-dependent  production,  and  oxygen-  b a c t e r i c i d a l mechanisms  during the respiratory burst,  include  of highly  toxic  oxygen derivatives such as superoxide anions,  hydrogen  hydroxyl r a d i c a l s ,  Oxygen-independent  and s i n g l e t oxygen (50).  mechanisms  comprise  functions.  These  a  include  binding proteins (13), certain  more  components  diverse  of  antimicrobial  lysosome a c i d i f i c a t i o n  production  of  group  of arginase  complement  (50),  peroxide,  (38),  (18), synthesis of  lysosomal  hydrolases  including lysozyme (7), and macrophage c a t i o n i c proteins In  recent  antibacterial  activity  several have  been  cationic  of  macrophage  c a t i o n i c proteins  rabbit alveolar macrophages appeared (36). MCP-1 are  and MCP-2, 33  In  from  1980,  (MCPs)  with several  the  first  isolated  from  These peptides, named  are r i c h i n arginine and cysteine  (37).  amino acids each i n length and d i f f e r by only  1  (36).  proteins  identified  types of phagocytes (Table 1).  different report  years,  iron-  a  They single  Table 1_. Examples of bactericidal cationic proteins of phagocytes. Source  Size  Human polymorphs- 37 kD nuclear leukocytes (PMNs)  Rabbit PMNs  Rabbit alveolar macrophages  References  Gram negative bacteria, 26,47,48 including Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa  57 kD  E. coli, S. typhimurium, Neisseria gonorrhoeae  48,52,55  55 kD  P. aeruginosa  15,16  25 kD  Staphylococcus aureus, E. coli  32,33  <3.5 kD  Rat PMNs  Activity  4-6 kD 50 kD  S. aureus, 5,6,9,43 E. coli, P. aeruginosa, Acinetobacter calcoaceticus (note: family of three distinct peptides) S. typhimurium, A. calcoaceticus  14,23  E. coli, S. typhimurium  4,53  4 kD  Gram negative and Gram positive bacteria, various fungi (note: family of six distinct peptides)  4 kD  (as above) (note: family of two distinct peptides)  2  substitution strength exhibit  (42).  Under  conditions of r e l a t i v e l y  and at near-neutral pH,  Gram p o s i t i v e and Gram negative organisms,  P.  of  various  fungal,  including P3eudomonas  (19,20,22,41).  aeruginosa,  being an opportunistic pathogen, i s of great  interest to this laboratory. the  Indeed,  accummulated evidence  mechanism of polycation i n t e r a c t i o n with the outer P_^ aeruginosa  l e d Hancock et a l (11) t o propose  promoted uptake model. and  ionic  MCPs have been demonstrated to  powerful antimicrobial e f f e c t s against  aeruginosa  low  aminoglycosides,  aeruginosa  by  on  membrane the s e l f -  That i s , polycations, such as polymyxins interact  displacing  with the outer  membrane  the divalent cations which  to  adjacent  Displacement  by large polycations r e s u l t s i n the disruption of and consequently,  agents l i k e lysozyme, thylamine, been outer  molecules.  increased permeability to  the hydrophobic fluorophor 1-N-phenylnaph-  and the ^-lactam,  proposed  (LPS)  serve  cross-link  the outer membrane,  lipopolysaocharide  of P.  that,  n i t r o c e f i n (10,11,12,24).  as a consequence of t h i s  membrane permeability,  I t has  perturbation  of  that the uptake of the polycation  i t s e l f i s promoted (11). The MCPs of  main objective of t h i s study was to determine  whether  behaved l i k e t y p i c a l polycations when mediating the k i l l i n g  P_^ aeruginosa.  To  test  this  hypothesis,  two  different  fluorescent probes were used, which have previously been shown to be e f f e c t i v e i n studying the interactions of polycations with aeruginosa  (24,27,28).  Furthermore,  3  P.  three d i f f e r e n t s t r a i n s of  P.  aeruginosa were used in order to examine possible differences  in  the interaction of MCPs with P_;_ aeruginosa  mutants.  These  strains were:  and  1)  H103:  PA01, wild type, previously described (30)  2)  H215:  PA01715, aminoglycoside super-sensitive, previously described (25)  3)  H234: AK1012, rough strain, previously described (17).  In this thesis, the results of a study on the interaction of macrophage  cationic  aeruginosa  are reported.  that  proteins  with  the outer  Evidence was obtained  membrane that  MCPs may function as an important mechanism of  k i l l i n g by macrophages.  4  of  P.  suggests bacterial  MATERIALS AND METHODS  Macrophage/granulocyte rabbits  were  Freund's weeks  injected  White,  rabbits  pentobarbital The  harvesting  female New Zealand  intravenously with  adjuvant (CFA) t o e l i c i t alveolar  later,  Ont.).  procurement.  were  (Euthanyl,  sacrificed  M.T.C.  1  ml  cells.  complete  macrophages. using  360  Pharmaceuticals,  lungs and trachea were then removed  of  of  Alveolar macrophages were  Three  mg  sodium  Mississauga, to  facilitate  collected  by  pulmonary lavage, introducing phosphate-buffered s a l i n e (PBS), pH 7.4,  v i a a tracheal cannula.  used  to  recover  peritoneal  cells.  exudates  Approximately 250 ml of buffer was To  harvest  granulocytes,  sterile  were obtained from 8-9 week old female  New  Zealand  white rabbits by i n t r a p e r i t o n e a l i n s t i l l a t i o n of 250  sterile  saline  containing  Chemical Company,  0.2$  S t . Louis,  glycogen  Mo.) and 5 ug LPS.  c o l l e c t e d approximately 16 hours l a t e r . as before, PBS. 10  (Type  VIII,  ml  Sigma  Exudates were  Rabbits were s a c r i f i c e d  and the peritoneum was lavaged with a t o t a l of 600 ml  C e l l s c o l l e c t e d either way were centrifuged at 200 x g f o r minutes  at  room  temperature and  washed  twice  with  PBS.  Contaminating erythrocytes, i f present, were removed by hypotonic lysis.  C e l l s were then counted i n a haemocytometer, and s l i d e s  were prepared with a cytocentrifuge (Cytospin 2, Shandon Southern Instruments, (Canlab,  Inc., Sewickley,  Vancouver,  B.C.)  Pa.) and stained with  to determine c e l l y i e l d and  5  Diff-quik purity.  Peptide  purification.  Selsted  et  al  (45)  Peptides were p u r i f i e d by the method with  modifications.  Washed  cells  of (10  8  macrophages or 10^ granulocytes) were resuspended i n 10 ml of 10$ acetic  acid  and  homogenized with a t i s s u e  minutes  at  4,000  rpm.  C e l l s were then  maximal disruption of c e l l membranes.  minutes.  fraction, 25  sonicated  for  3  to  ensure  To remove unbroken  cells  the homogenate was centrifuged at 250 x g at 4 C f o r  and n u c l e i , 10  homogenizer  The  was  supernatant,  i . e . , the  lysosome-enriched  then extracted f o r 4-6 hours i n the  presence  ug/ml Pepstatin A (Sigma Chemical Comp.) at 4 C.  of  Lysosomes  and c e l l u l a r debris were then removed by centrifugation at 27,000 x  g.  acid  The p e l l e t was reextracted overnight i n fresh 10$ to ensure complete recovery of peptides.  supematants for  48  aoetic  The 27,000  x  g  were then dialyzed against 0.1$ acetic acid at 4  C  hours i n Spectrapor 3 (MW c u t o f f 3,500) d i a l y s i s  tubing  (Spectrum Medical Industries, Inc., Los Angeles, Ca.). Following dialysis, The  the supematants were concentrated by  lyophilized  retentate  was referred to as  which was used i n some experiments.  lyophilization. "crude  extract"  Approximately 10 mg of crude  extract was resusupended i n 1 ml of 1$ acetic acid and applied to a  column  (2.5 by 30 cm) containing Bio-Gel  P-10,  50-100  mesh  (BioRad Laboratories, Richmond, Ca) that had been equilibrated at room  temperature i n 1$ acetic a c i d .  constant collected.  flow  rate of 5 ml per hour,  The  effluent  was  Proteins were eluted at and 1 ml  monitored  by  absorbance at 214 and 280 nm i n a model PU8600 (Pye Unicam Ltd., Cambridge,  Eng.).  6  a  fraotions  were  measuring  its  spectrophotometer  Samples of fractions were  also examined on acid-urea slab g e l s , run by the method of Panyim and  Chalkley  weight  (35).  basio  Fractions containing  peptides  collected,  pooled,  separation  of  (MCPs  and  or  low  molecular  neutrophil peptides)  lyophilized.  Final  were  purification  and  the peptides was achieved by reversed-phase  Protein Liquid Chromatography (Pharmacia, a Pro-RPC 5/10 column. 0.1%  only  trifluoroacetic  concentration  was  Uppsala, Sweden) using  Water-acetonitrile gradients acid  were  Fast  used  in  assessed by the BioRad  containing  elution.  Protein  Protein  Assay  using  Standard I (Bovine Plasma Gamma Globulin).  B a c t e r i a l s t r a i n s and growth conditions. PA01  strain  H103 was used i n  Pseudomonas aeruginosa  a l l experiments.  A  gentamioin  super-sensitive s t r a i n (H215) and a rough mutant (H234) were used for  certain  experiments.  proteose  peptone  no.  2 medium  (Difco  Mich.).  Experimental  cultures  were started from an  broth c u l t u r e ,  diluted 1:50  1%  A l l s t r a i n s were grown i n Laboratories,  (w/v)  Detroit, overnight  i n t o fresh medium, and grown at 37 C  (or 30 C f o r H234) with vigorous shaking to an o p t i c a l density of 0.4 to 0.6 at 600  Antibacterial  nm.  assay.  The  bactericidal  assay  exactly as described by Lenrer et. al_ (20). washed Assay colony  three  forming  (control),  10  Briefly,  times i n 10 mM sodium phosphate  mixtures,  was  buffer,  performed o e l l s were 7.^.  pH  i n a t o t a l volume of 100 u l , consisted of units ug  in  buffer,  crude extraot,  7  to  which  distilled  105  water  or 5 ug p u r i f i e d peptide  was  added.  After incubation a t 37 C f o r 60 minutes, s e r i a l d i l u t i o n s  were made i n the same buffer and v i a b l e counts determined.  LPS  isolation.  Hancock (3).  LPS was isolated as desoribed by Darveau and The i s o l a t e d LPS was extracted twice with an equal  volume  of ehloroform-methanol (2:1) t o remove trace  sodium  dodecyl s u l f a t e and phospholipids which resulted from the  isolation  procedure  (3).  amounts of  Residual chloroform was  removed  by  purging with nitrogen gas i n a fume hood f o r about 30 minutes.  A  10 mg/ml stock suspension of H103 and H215 LPS was prepared on a dry weight b a s i s .  Dansyl-polymyxin binding experiments. prepared  as described  by  Dansyl-polymyxin (DPX) was  Sohindler  by dinitrophenylation ( 1 ) .  quantitated  (39)  and Teuber  and  DPX binding t o LPS or  whole c e l l s was monitored by measuring the fluorescence i n t e n s i t y on  a model 650-10S fluorescenoe spectrophotometer  (The Perkin-  Elmer Corp., Norwalk, Conn.) set with an excitation wavelength of 340 nm and an emission wavelength of 485 nm with s l i t widths o f 5 nm.  Binding  (27).  assays were performed as described by Moore et a l  Briefly,  containing  3  aliquots  ug/ml  of DPX were t i t r a t e d into a  (0.3 uM) LPS i n 1 ml of 5  hydroxyethyl-piperazine-N'-2-ethanesulfonic and  strip  determined f 0  HEPES (N-2-  acid) buffer, pH 7.2,  the fluorescence i n t e n s i t y was recorded on a  Coleman  (  mM  /F  m a x  )  chart  by using x  recorder.  The amount of DPX  the equation:  concentration  of DPX,  8  cuvette  amount where f  Perkin-Elmer bound  of DPX Q  i s the  was  bound  =  observed  fluorescence  at  (subsaturating  a given DPX LPS  concentration added to 3 ug/ml  concentration)  and  F  is  the  LPS  observed  ID £UC  fluorescence excess  when (300  LPS  experiments, DPX 5  mM  the  HEPES  (27).  ug/ml)  10  with  7.2,  pH  For  is  binding  to  added  whole  to cell  t i t r a t e d i n t o a cuvette containing 9 9 0 u l of  was  buffer  respiration),  same concentration of DPX  mM ul  10  and  sodium of  azide  cells  (to  inhibit  resuspended  to  an o p t i c a l density at 6 0 0 nm of 0 . 5 i n the same b u f f e r . Binding i n h i b i t i o n experiments.  I n h i b i t i o n of DPX  was  performed as previously described  of  DPX  binding,  including MCPs,  containing  3  saturation  of the LPS by the DPX)  7.2,  pH  Briefly, inhibitors  were t i t r a t e d i n t o  ug/ml LPS and 2 . 5 uM DPX  a  (resulting i n 8 5  i n 1 ml of 5 mM  HEPES  and the decrease i n the observed fluorescenoe  i n h i b i t i o n ) was was  (27).  binding to LPS  recorded.  calculated  as  inhibition  intercept  gave  inhibitor  giving  concentrations  -1/I  50%  ,  where  was  the  maximal i n h i b i t i o n at  a  the  LPS  binding to whole o e l l s ,  an  o p t i c a l density of 0 . 5 at 6 0 0 nm i n the same b u f f e r ,  azide,  pH 7 . 2 ,  1 0 u l of  HEPES  mM  concentration required to give  inhibition  and  The  and  inhibitors  10  DPX  plot  concentration  i n t o a cuvette containing 9 9 0 u l of 5 mM sodium  (peroent  of x of DPX  used.  For i n h i b i t i o n of DPX titrated  buffer,  versus 1 / i n h i b i t o r concentration. 5 Q  90$  to  Maximum i n h i b i t i o n by a given compound  the extrapolated y intercept of  1/percent  cuvette  I  5 Q  values  85-90$  cells  were read o f f of  9  buffer/  resuspended  saturation. a  graph  were  and  to the  Maximal of  the  I n h i b i t i o n curves. Permeabilization Uptake  of whole  cells  described  (24).  Briefly,  washing twice i n 5 mM HEPES buffer, (to i n h i b i t r e s p i r a t i o n ) , 0.5  then  1-N-phenylnaphthylamine.  of 1-N-phenylnaphthylamine (NPN) assays were performed as  previously  of  to  c e l l s were  pH 7.2,  before  NPN  (Sigma) was  density  The c e l l suspension  to s i t at room temperature f o r 30  use.  by  containing 1 mM KCN  and resuspended to an o p t i c a l  a t 600 nm i n the same b u f f e r .  allowed  prepared  dissolved  to 60  was  minutes  i n acetone  at a  concentration of 500 uM and added to 1 ml of c e l l suspension to a f i n a l concentration of 10 uM.  MCPs and crude macrophage extract  were tested f o r the a b i l i t y to permeabilize c e l l s to NPN, and the increase  in  monitored.  NPN  fluorescence i n t e n s i t y  Excitation  was  continuously  and emission wavelengths were s e t at 350  and 420 nm, respectively, with s l i t widths of 5 nm. To determine the e f f e c t s of pH on enhancement of NPN by MCPs,  a separate experiment was done i n which b a c t e r i a l c e l l s  were prepared as before,  except that o e l l s were resuspended i n 5  mM HEPES buffer plus 1 mM KCN at pH 5.5, 8.0, before  uptake  and  allowed  use.  The  6.0,  6.5, 7.0, 7.5, or  to s i t 30 to 60 minutes at room  temperature  permeability assay was then done  exactly  as  described.  Enhancement assay  was  of phagocytosis by MCPs. done  The  as previously desoribed by  (in press) with some modifications.  10  phagocytosis  visual  Battershill  et a l  Unelicited rabbit  alveolar  macrophages were c o l l e c t e d and washed as before, to  5  x  10  supplemented  5  cells/ml i n  RPMI-1640  (Gibco,  with 44 mM sodium bicarbonate  and  resuspended  Burlington, (Fisher  Ont.)  Scientific,  Vancouver, B.C.), 10$ (v/v) f e t a l c a l f serum (Gibco), 10 mM HEPES buffer (Terochem Laboratories, mercaptoethanol  (BioRad),  Vancouver,  B.C.), 0.04$ (v/v) 2-  2 mM L-glutamine  (Sigma), 40 units per  ml p e n i c i l l i n and 40 ug/ml streptomycin (Giboo), aliquots  of  the  pH 7.2.  o e l l suspension were incubated i n 35 x  Two  ml  10  mm  NuncIon tissue culture dishes at 37 C i n 10$ COg overnight. prior  to the assay,  twice  with  the macrophage monolayer was gently  phagocytosis  assay  medium  supplemented with 10 mM HEPES buffer only), medium  was placed over the monolayer.  resuspended  MCP-1  1640  washed medium  then one ml of  this  Bacteria were washed  and  to 10° c e l l s / m l , and added t o the monolayer to give a  f i n a l bacteria to macrophage r a t i o of 20:1. of  (RPMI  Just  Equal concentrations  and MCP-2 were added to the system to a f i n a l  peptide  concentration of 50 ug/ml or an equal volume of s t e r i l e water was added (control). in dish were  10$ COg,  The system was incubated for 90 minutes at 37 C  at which time the macrophages were scraped from the  using a rubber policeman and gently  resuspended.  prepared by cytocentrifugation of 100 u l aliquots  by s t a i n i n g .  To assess phagocytosis,  Slides followed  the number of bacteria i n  each of 120 macrophages was recorded by v i s u a l inspection.  11  RESULTS  Purification of MCPs.  Peptides were originally sought from three  macrophage or macrophage-like c e l l lines,  inoluding:  P388D.J, a  DBA/2 murine macrophage tumour c e l l line; PU51.8, a BALB/c murine macrophage-like lymphocyte  tumour  fusion  cell  line;  and  DC7,  a macrophage-T  produot  with macrophage properties, such as  adherence and phagocytosis.  However, after extensive screening,  no small basic peptides were found in any of the c e l l lines used, as  judged by the lack of proteins migrating more cathodal  than  lysozyme  on acid-urea gels (data not shown).  cationic  proteins from rodent phagocytes have been described  rabbits,  guinea pigs and rats,  no  To date,  but not mice (Table 1).  peptides were present in any of the c e l l lines used,  small  Because it  decided to turn to the original source of macrophages from MCPs  had  originally  macrophages. granulocytes  Later, also  been it  -2  (45).  was  learned  ie, that  rabbit rabbit  was which  alveolar peritoneal  produced peptides identical in structure  function to MCP-1 and -2, and  isolated,  called NP (for neutrophil peptide)  As granulocytes were  faster  and  in  more  and -1  easily  harvested, much of the peptides used in this report were obtained from these c e l l s . high. of  Obtained from either source,  c e l l purity was  From CFA-elicited rabbits, which were shown to yield cells higher  purity  with  increased  12  peptide  content  (21),  approximately  10  alveolar macrophages could be recovered  >98$  p u r i t y and v i a b i l i t y (as judged by trypan blue  and  from  sterile  granulocytes  peritoneal >96$  of  exudates,  purity  were  with  exclusion);  approximately routinely  10^  recovered.  Electrophoresis on acid-urea gels of extracts prepared from these sources  revealed  the  presence  of  two  bands  migrating  past  lysozyme (which were not present i n c e l l l i n e preparations), i e , MCPs. were  In the o r i g i n a l protocol (42), used  to  strips.  preparative acid-urea gels  i s o l a t e the peptides by elution  However,  recovering  the  of  excised  peptide free of  5  gel  M  urea  proved time-consuming and wasteful. A l t e r n a t i v e l y , g e l f i l t r a t i o n was  used.  A  separating or  six  single  column  run  in  purification  the  sample ( F i g . 1).  approximately 70%,  differential  larger  effective  macrophages  molecular  The f i n a l  weight  separation  Peptide recovery was  and  with small losses  centrifugation,  incomplete  estimated  occurring  extraction,  due  preparation yielded approximately 200 pg of each of the peptide from 10  Antibacterial  8  Each purified  Q alveolar macrophages or 10* granulocytes.  assay.  MCPs  were  previously found to  be  under conditions of r e l a t i v e l y low ionic strength  near-neutral pH  to  molecular  s i e v i n g , and monitoring samples during the p u r i f i c a t i o n .  effective  in  of the peptides was achieved by Fast Protein Liquid  Chromatography (FPLC) ( F i g . 2). be  most  the desired family of peptides (two from  from granulocytes) from other  proteins  to  proved  (20),  hence,  the buffer  used.  Although  e f f e c t s of k i l l i n g were not as dramatic as those seen with  13  most and the other  Figure from 1$  1_.  P-10 column f r a c t i o n a t i o n .  granulocytes was applied to the column equilibrated with acetic acid.  One ml f r a c t i o n s were c o l l e c t e d ,  analyzed f o r protein content. are  Ten mg of crude extract  identical  to MCP-1  and  and  samples  Neutrophil peptide (NP) -1 and -2,  respectively.  Samples  -2  were  electrophoresed on an acid-urea polyacrylamide g e l containing 15$ acrylamide and 5 M urea.  Symbols:  CE, 100 ug crude granulocyte extract.  14  L,  hen egg white lysozyme;  60 75  L  CE  Figure  2_.  MCP  purification  by  reversed  phase  FPLC.  Approximately 250 ug pooled neutrophil peptides were applied to a Pro-RPC  5/10  containing ml/min  was  column.  A  linear  water-acetonitrile  gradient  0.1$ (v/v) t r i f l u o r o a c e t i c acid a t a flow rate of 0.3 used to elute peptides.  c o l l e c t e d and read  Fractions  spectrophotometrically.  16  (0.6  ml)  were  7  9  11  13  Fraction number  17  15  b a c t e r i a l species,  found that the rough s t r a i n (H23M  i t was  more susceptible to the k i l l i n g than  the  e f f e c t s of MCPs and crude extract  wild type smooth s t r a i n H103  sensitive  strain  H215  was  (Table  2).  or the gentamicin MCP-1  was  super-  slightly  more  b a c t e r i c i d a l than MCP-2 f o r a l l s t r a i n s tested. Despite killing bacteria  the  assay, and  crude extract being very no  additional k i l l i n g  peptide were co-incubated  efficacious  e f f e c t s were with 20  in  the  seen  ug/ml  when  hen  egg  white lysozyme (data not shown).  DPX  binding to p u r i f i e d LPS.  DPX  binds to p u r i f i e d LPS r e s u l t i n g  i n enhancement of fluorescence (27). a  When DPX  was t i t r a t e d i n t o  s o l u t i o n containing LPS, an increase i n fluorescence was  u n t i l a l l of the s i t e s which bind DPX  seen  on the LPS were f i l l e d ( F i g .  3,  r e f . 27).  However, H23M LPS would not saturate, even when up  to  12  was added and consequently  uM DPX  experiments.  The  data  f o r H103  analyzed on a H i l l p l o t ( F i g . 4). was  a  and H215  LPS-DPX  H i l l number,  in  these  binding  was  The slope of the H i l l p l o t  s t r a i g h t l i n e with a slope greater than  cooperative i n t e r a c t i o n of DPX  with LPS.  one,  (n)  suggesting  The value for n was  the  which provided a minimal estimate of the number  cooperative i n t e r a c t i o n s i t e s . higher  was not used  than  that  f o r H103  The H i l l number f o r H215  LPS  (Table  3),  of  LPS  suggesting  was  higher  oooperativity which might have resulted from the presence of more DPX  binding s i t e s .  measure strains.  of  The x intercept of the H i l l plot ( S  the a f f i n i t y of LPS for DPX, The  information  5  )  were s i m i l a r f o r  derived from these  18  Q  experiments  f  a  both was  Table  2.  K i l l i n g of P_j_ aeruginosa s t r a i n s by crude macrophage  extract and p u r i f i e d peptides.  The k i l l i n g assay was  performed  exactly as described by Lehrer et a l (20), exposing approximately 10^ bacteria f o r 60 minutes to 100 ug/ml crude extract, p u r i f i e d peptide, or s t e r i l e d i s t i l l e d water (control).  19  50 ug/ml  % SURVIVORS  Strain  Control  Crude Extract  MCP-1  MCP-2  H103  100  10  19  28  H215  100  3  19  22  H234  100  <1  1  5  20  Figure  3_.  LPS-dependent  fluorescence  of  dansyl-polymyxin.  Dansyl-polymyxin was t i t r a t e d i n t o a cuvette containing 1 ml of 5 mM  HEPES buffer,  Symbols:  •  pH 7.2,  with or without 3 ug/ml (0.3 uM) LPS.  , s t r a i n H103 LPS; O , s t r a i n H215 LPS; x , no LPS.  21  Figure 4_. were  derived  fluorescence the  H i l l plot of dansyl-polymyxin binding to LPS. Values from  data i n F i g .  3,  where f  at a given dansyl-polymyxin concentration and F i s  maximum fluorescence (extrapolated from the graph) when  i s completely saturated with dansyl-polymyxin, lack is  i s the observed  LPS  as revealed by a  of further fluorescence increase when more dansyl-polymyxin added.  independent linear  Data  (shown  experiments.  f o r H215)  are averages  The c o r r e l a t i o n c o e f f i c i e n t  of  f o r the  regression of the data for t h i s and a l l other H i l l  was greater than 0.99.  23  three  plots  3..  Table  aeruginosa Hill  plots  Kinetics strain  of binding  H103 and H215 LPS.  (eg. F i g . 4 ) .  dansyl-polymyxin  at  The S  Q  to  Values are derived i s the concentration  P. from of  which one h a l f of the binding s i t e s on the  LPS molecule are saturated. degree  of dansyl-polymyxin  The H i l l c o e f f i c i e n t indicates the  of binding c o o p e r a t i v i t y .  The maximum number of binding  s i t e s was calculated as amount dansyl-polymyxin divided by number of LPS moleoules present  (27).  25  H i l l coefficient Strain  S  (uM)  (n)  Maximum no. of binding s i t e s / LPS molecule  H103  1.02  2.01  4.32  H215  1.12  3.53  5.21  26  useful f o r i n t e r p r e t i n g DPX i n h i b i t i o n r e s u l t s .  Inhibition the  of DPX binding t o LPS by polycations.  self-promoted  permeability  uptake hypothesis,  Acoording  polycations  b a r r i e r by d i s p l a c i n g M g  2+  disrupt  which serves  to the  t o cross-  l i n k adjacent LPS molecules on the surface of the outer membrane. Further,  certain  shown  t o be  (27).  MCPs  for  polycationic  compounds have  previously been  capable of competing with DPX f o r binding  to  LPS  were a l s o found to be capable of competing with DPX  binding t o LPS to some extent  displacement  (Fig. 5).  The  competitive  of DPX by MCPs suggested that at least some of the  binding s i t e s on LPS which bound DPX were also capable of binding MCPs.  Similar displacement was observed using gentamicin or M g  as i n h i b i t o r s (Table 4 ) .  2+  From the graph, i t might appear that  MCP-1 displaced DPX from H103 LPS more e f f e c t i v e l y than from H215 LPS.  However,  displaced binding  H103  MCP-1  was  to d i s p l a c e h a l f the maximal p o s s i b l e i n h i b i t i b l e  DPX  H215 LPS (a lower I  from  affinity MCP-2. amenable  i t s LPS f o r DPX.  5Q  f o r MCP-1 As the to  t o H215 LPS.  value), Further,  value),  H215  was more  on  LPS (a higher MI  DPX had  sites  required bound  from  i t should be noted that although more  less  indicating  a  higher  Similar r e s u l t s were seen with  i n h i b i t i o n curve f o r MCP-2 and H215 LPS was similar  analysis  (inhibition  appeared  from  be  cooperative),  the values  graph i n F i g .  5, panel B, and must not be considered d e f i n i t i v e .  The  reported were extrapolated  to  not  the  data indicated that MCPs were very e f f i c i e n t competitors f o r  LPS binding, as indicated by the low I  27  R  n  values (Table 4 ) .  Figure  5_.  Inhibition  of dansyl-polymyxin binding t o LPS by  MCPs.  MCPs were t i t r a t e d i n t o a cuvette containing 3 ug/ml (0.3  uM) LPS and 2.5 uM dansyl-polymyxin i n 1 ml of 5 mM HEPES buffer, pH 7.2, •  and the decrease i n fluorescence was recorded.  , s t r a i n H103 LPS; O , s t r a i n H215 LPS.  28  Symbols:  % Fluorescence o  VO  o  o  o  o  o  Table 4.*  I n h i b i t i o n by various polycations of  binding to P. aeruginosa s t r a i n H103 MI  and H215  dansyl-polymyxin  LPS.  The I  5U C A  and  values were determined as described i n Materials and Methods,  except  for  H215  LPS and MCP-2,  graph ( F i g . 5, panel B).  30  which was  extrapolated  from  a  H103  Inhibitor  I  5  Q  H215  Maximal Inhibition  I  5  Q  Maximal Inhibition  MCP-1  2.30 uM  62.5$  1.63 uM  45.5$  MCP-2  2.98  42.7  1.75  25.0  polymyxin  6.10  gentamicin Mg  2+  71.9 1,840  100  8.20  42.7 45.5  3  1  60.6 1,900  100 44.8 46.9  DPX  binding and I n h i b i t i o n to whole o e l l s .  To show  could Interact with LPS i n native outer membranes, DPX  binding  was  studied using i n t a c t c e l l s .  that  MCPs  I n h i b i t i o n of  Because  of  the  difference i n binding curves for each of the s t r a i n s ( F i g . 6) and the  complexity of i n h i b i t i o n curves f o r some of the  for  some s t r a i n s ,  off  of graphs,  and MI values (Table 5) were read d i r e c t l y such as the one i n Figure 7.  LPS, MCPs were capable of d i s p l a c i n g DPX H103.  However,  H215  oells,  showing that i n intaot c e l l s ,  strain  readily  more  greater displacement  purified  of DPX  H215  ocourred  from  MCPs interacted  r e a d i l y than with H103  removed from H234.  As with  from whole c e l l s of  and  this  polycations  cells.  DPX  with  was  not  Recalling that H23 * was best k i l l e d 1  by the peptides, i t i s probable that t h i s r e f l e c t s the r e l a t i v e l y higher a f f i n i t y f o r the probe DPX  than f o r MCPs.  Enhancement of NPN uptake by MCPs. to  be  an  excellent  aminoglycosides to  the  probe  with  study  the  interactions  of  aeruginosa, as uptake was presumably due  permeabilization  a n t i b i o t i c (24).  to  NPN had previously been shown  of  the outer membrane  by  the  added  A s e r i e s of experiments were performed i n which  various concentrations of p u r i f i e d peptides or crude extraot were added  to  cyanide-pretreated  fluorescence increase, plot  of  cells,  and the i n i t i a l  r e f l e c t i n g NPN uptake,  8,  were recorded.  the i n i t i a l rates of fluorescence increase  concentration of peptide or crude extract, yielded  a  sigmoidal  curve  32  rates  of A  versus  the  such as that i n  Fig.  i n a l l cases.  Such  a  curve  Figure 6. polymyxin HEPES washed  Binding of dansyl-polymyxin was  titrated  buffer/10 twice  Dansyl-  i n t o a cuvette containing 1 ml of  mM sodium azide,  and resuspended  density of 0.5 a t 600 nm.  to i n t a c t c e l l s .  pH 7.2,  and 10 u l of  i n the same buffer to an  Symbols: •  c e l l s ; O , H234 c e l l s .  33  , H103 c e l l s ;  O  5  mM  cells  optical , H215  Table binding  5_. to  inhibition  Inhibition whole  by various polycations of dansyl-polymyxin aeruginosa c e l l s .  I^Q  and  maximal  (MI) values were read d i r e c t l y o f f of graphs,  such as  that i n F i g . 7.  35  The  H234  H215  H103  Inhibitor  I...  polymyxin  0.55 uM  55$  0.29 uM  76$  0.80 uM 60$  MCP-2  2.50  30  1.13  65  2.50  5  MCP-1  2.81  50  2.25  54  5.63  15  gentamicin Mg  2+  MI  I,..  MI  I  c  n  MI  36  50  20  70  40  55  2,400  75  1,200  84  2,000  80  36  Figure 7_. by MCPs.  I n h i b i t i o n of dansyl-polymyxin binding t o whole c e l l s MCPs were t i t r a t e d  i n t o a cuvette containing 1 ml of 5  mM HEPES buffer/10 mM sodium azide, pH 7.2, 10 p i of c e l l s washed twice and resuspended to an o p t i c a l density of 0.5 at 600 nm, and the  dansyl-polymyxin  saturation and H234). cells;  O  concentration  required  to cause  of whole c e l l s (1.87 uM f o r H215 or 3.75 uM f o r Symbols: •  , s t r a i n H103 c e l l s ;  , s t r a i n H234 c e l l s .  37  O , strain  85-90$ H103 H215  Figure  8^  Enhancement of NPN uptake by  P_j_ aeruginosa  cells.  Varying  cuvette  containing 1 ml of c e l l s ,  optical  density of 0.5 at 600 nm i n 5 mM HEPES buffer/1 mM  pH  7.2,  and  10  concentrations  intact  uM NPN.  of MCP-1 were t i t r a t e d  H103 c e l l s ; O , H215 c e l l s .  39  a  washed and resuspended to an  I n i t i a l rate of uptake  r e f l e c t e d by fluorescence increase,  into  were recorded.  of  NPN,  KCN, as  Symbols: • ,  INITIAL RATE  suggested  positive cooperativity.  obtained f o r both H103 and H215, occurred  Further,  s i m i l a r curves were  but uptake of the probe by H215  at s l i g h t l y lower concentrations,  suggesting that H215  was s l i g h t l y more e a s i l y permeabilized than was H103. not  be used i n t h i s assay,  membrane. numbers  When  were  cooperative  the data was reanalyzed by  obtained  plots,  Hill  number  of  These numbers were s i m i l a r f o r both  MCP-1 was a better permeabilizer than MCP-2,  f o r both s t r a i n s .  The S  Q  ug/ml,  values were less f o r  i n d i c a t i n g a higher a f f i n i t y of MCP-1 f o r the c e l l s  MCP-2. However, 20  Hill  outer  maximal uptake of NPN at 40 ug/ml r e l a t i v e to 70  respectively, MCP-1,  stable  whioh indicated the minimal  binding s i t e s .  s t r a i n s (Table 6). causing  perhaps due to a less  H234 could  than  maximal uptake f o r both strains occurred at only  ug/ml when crude macrophage extract was used,  i n d i c a t i n g the  possible presenoe of other lysosomal contents with permeabilizing activity. The were  most  observed  i n t e r e s t i n g r e s u l t s obtained using the NPN  assay  when  oells  the same assay was  resuspended i n buffer of varying pH. pH  from 5.5 to 8.0,  conducted  using  The buffers used ranged i n  the presumed range of lysosomal pH  (2,40).  At pH 7.5 and 8.0, MCP-1 exhibited r e l a t i v e l y poor permeabilizing aotivity.  However, below pH 7.0, the peptides caused remarkable  permeabilization,  with  addition  of  ug/ml MCP-1 or less  however,  the e f f e c t s of MCP-1 addition could not be observed  the  cells  7.5  reached  peptide a d d i t i o n .  maximal  NPN uptake occurring (Fig. 9 ) .  with  the  pH  5.5,  At  maximal fluorescence i n the absence  as  of any  This was presumably due to the protonation of  41  Table 6. P.  H i l l numbers and S 'a  aeruginosa.  Q5  Values  f o r interactions of MCPs  with  were derived from H i l l plots drawn from  p l o t s of i n i t i a l rate of uptake versus MCP concentration, suoh as the one i n F i g .  8.  A H i l l number with a value of greater  one indicates p o s i t i v e cooperativity.  42  than  MCP-1 S  MCP-2  Strain  n  H103  2.77  5.00 uM  1.15  24.55 uM  H215  3.00  4.26  1.46  17.7  Q > 5  43  n  S  q  %  5  Figure  9_.  intact  P_^ aeruginosa  washed  and  shown.  The assay was then performed exactly as before.  pH 6.0;  MCP-1  promoted enhancement of NPN s t r a i n H103 at varying  resuspended i n 5 mM HEPES buffer/1  fluorescence i n pH. mM  Cells KCN,  were pH  as  Curve a,  curve b, pH 6.5; curve c, pH 7.0; curve d, pH 7.5; curve  e, pH 8.0.  44  phosphate  groups i n LPS,  r e s u l t i n g In decreased  cross-bridging  and hence, a decrease i n outer membrane s t a b i l i t y .  Enhancement of phagocytosis.  When b a c t e r i a and macrophages were  co-incubated i n the presence of peptides, s i g n i f i c a n t enhancement of  phagocytosis  by  unelicited  rabbit  alveolar  occurred f o r a l l b a c t e r i a l s t r a i n s (Table 7). noted  that  uptake of the rough s t r a i n  macrophages  I t should also be was  significantly  higher than that of H 1 0 3 and H215 i n the absence of peptide.  46  Table  £.  aeruginosa  Phagocytosis by  bacteria  per  presence  of  (control). two-tailed  of  untreated  and  u n e l i c i t e d rabbit alveolar  MCP-ooincubated macrophages.  macrophage were incubated f o r 90  minutes  50  distilled  ug/ml  Values student's  peptides  or  sterile  shown are means + standard t  test,  phagocytosis  enhanced i n the presence of MCPs (p<0.002).  47  P.  Twenty i n the  deviations.  water By  i s significantly  Number of bacteria phagocytosed per macrophage Strain  MCP-coincubated  Control  H103  2.59 +  1.62  5.59 +  2.84  H215  3.53  +  2.32  5.54 +  3.16  H234  6.25 +  2.94  8.81 +  4.82  48  DISCUSSION  Polycationio  antibiotics,  aminoglycosides,  have  such  as  polymyxins  and  been shown to cause enhancement of  outer  membrane permeability of P. aeruginosa (12,21,30). these  polycations  The uptake of  has been hypothesized to occur v i a the  promoted uptake pathway,  i n which polycations d i s p l a c e  self-  divalent  cations from s i t e s where they noncovalently cross-bridge adjacent lipopolysaccharide molecules (30,31). outer  membrane  i s disrupted,  l i k e lysozyme and NPN  c e l l s become permeable to  polycationio,  typical  enhanced  i t was found that  of other known permeabilizers (12).  first  MCPs,  the uptake of NPN i n Further,  shown to interact with high a f f i n i t y to the LPS of P^ (the  agents  (12,24).  In accord with t h i s theory, strongly  Once the i n t e g r i t y of the  requirement of the self-promoted uptake  being  a  manner  MCPs were aeruginosa  model),  as  demonstrated by competitive displacement of DPX bound to p u r i f i e d LPS or whole c e l l s (Tables 4 and 5). the  displacement of DPX  from H234 whole c e l l s ,  poorly displaced ( F i g . 7). six  groups  in  unpublished  data).  strain  l o s t i t s MCP  had  smooth  Thus,  i n which DPX  was was  Strain H234 i s known to possess only  phosphate groups per LPS molecule,  phosphate  The exception to this  strain  compared with 12 LPS  (E.J.  to  MoGroarty,  i t i s p o s s i b l e that the LPS of  binding s i t e (or that the  affinity  H234 LPS f o r MCPs was decreased r e l a t i v e to i t s a f f i n i t y f o r  49  15  this of the  probe DPX). might  be  The greater s u s c e p t i b i l i t y of H23 * to MCPs (Table 2) 1  due  to a generally less stable  outer  membrane  (see  Hancock, r e f . 10) as suggested for other rough s t r a i n s . MCPs  did  not  mediate c e l l u l a r l y s i s i n  lysozyme at the concentrations tested, with  lysozyme  shown). capable  of  membrane,  was a t y p i c a l ,  protected  the  in  in  cell  lysis  that  MW  1000)  the  rabbit lysozyme,  for  the  (12).  action  A  sterio  not  outer  possible hindrance The  have been used i n the lysozyme l y s i s  assay  molecules  of  (eg.  r e l a t i v e to MCPs (MW  gentamioin, 4000).  egg white lysozyme was used i n these assays.  Thirdly,  (data  lysozyme.  general smaller  polymyxin,  cellular  from  of  as many polycationic compounds are  f o r t h i s phenomenon could be that  permeabilizers were  effect  enhancing the passage of lysozyme across resulting  explanation  presence  nor did the co-incubation  i n the k i l l i n g assay have any  This  the  MW  500;  Secondly,  hen  I t i s possible that  had i t been a v a i l a b l e , may have been e f f e c t i v e .  there was a l i m i t a t i o n i n the amounts of MCPs a v a i l a b l e  these  studies,  and  the lysozyme l y s i s assay i s  known  to  require much larger amounts of permeabilizer, such that an effect might have been seen at higher concentrations. the  effectiveness  permeabilization other  lysosomal  synergistic, role  of  the  of c e l l s ,  crude extract  in  i t i s l i k e l y that  constituents,  take  role i n bacterial k i l l i n g .  an  However, the  due to  killing  lysozyme, active,  and  and/or  possibly  On the other hand, the  of lysozyme i n the phagolysosome may be purely degradative. It  was  clear  from the DPX  and NPN  experiments  surface of the b a c t e r i a l o e l l was somehow being  50  that  modified,  the since  the  fluorescence  intensity  of the probes changed i n  dependent on t h e i r environment (Figs.  5,7, and 8).  a  manner  Furthermore,  preliminary electron microscopic i n v e s t i g a t i o n s (in c o l l a b o r a t i o n with  Nancy Martin) revealed surface changes of MCP-treated c e l l s  compared  with  phagocytosis macrophages  untreated of  also  cells.  MCP-coincubated suggested  Finally, cells  enhancement  by  rabbit  that surface changes  of  alveolar  had  occurred.  The p o s s i b i l i t y was raised that these changes could have been the result  of  inoreased  (D.P.Speert,  personal  hydrophobicity  due  communication).  to To  MCP  interaction  investigate  this  p o s s i b i l i t y , p a r t i t i o n i n g of MCP-treated and untreated c e l l s i n a biphasic system of polyethylene g l y c o l and dextran was However,  no  s i g n i f i c a n t changes i n p a r t i t i o n i n g behaviour  seen (data not shown). was  that  phase,  due  any  to the negatively-charged nature of  the  this  dextran  increase i n surface hydrophobicity was abrogated However,  or none of the hydrophobic probe NPN was MCP  were  The most p l a u s i b l e explanation f o r  i o n i c i n t e r a c t i o n s with dextran.  before  attempted.  the fact that l i t t l e  taken up by whole  addition i s strongly i n favour of Increased  hydrophobicity  due  to MCP  by  i n t e r a c t i o n with the  cells  surface  bacterial  cell  surface. Indeed,  the r o l e of hydrophobic i n t e r a c t i o n s between  phagocyte b a c t e r i c i d a l proteins and the surface of typhimurium has been documented (46,51,55). investigated  the  role  of  LPS  chain  finding  s u s c e p t i b i l i t y of Gram negative bacteria to various proteins  increased  in  Inverse proportion  51  c o l i and  Other studies  length,  to  other S.  have that  bactericidal  0-antigen  chain  length, their  and  that  isogenic  deep rough mutants are more susceptible  smooth s t r a i n  parents  (34,48,49,51,54).  than This  conclusion can now be extended to include the i n t e r a c t i o n of MCPs with  P_^ aeruginosa,  as H234 was indeed more susceptible to the  b a c t e r i c i d a l e f f e c t s of these peptides Hypotheses mutants  increased  susceptibility  t o b a c t e r i c i a l proteins have previously  For example, chain,  would  (46,48,52,55). would  of LPS  been  proposed.  decreased length of core, or the absence of O-side  electrostatic  LPS  to explain  (Table 2 ) .  tend  to expose l i p i d A,  interactions  with  making  hydrophobic and  the proteins  A l t e r n a t i v e l y , defects i n M g  d e s t a b i l i z e the outer membrane  2+  more  likely  binding by rough  (10), r e s u l t i n g i n  increased s u s c e p t i b i l i t y to b a c t e r i c i d a l proteins. The  most  finding  that  interesting  were  aeruginosa occurring  previously between  9).  acidification  shown to exhibit maximal  as  cells,  (PMNs),  are g r e a t l y  of P.  pH 7.0 and 8.0, with l i t t l e or no  killing  i t may  define  acidification  Although this  the r o l e  of the lysosome.  of MCPs  Initially,  during  MCPs could k i l l ingested  proceeds,  may  the granules several  appear  during  the  the b r i e f baoteria.  MCPs could permeabilize  making them more susceptible t o other macrophage  systems, or to degradative In  was i n  killing  transient r i s e i n pH (2,40), Then,  study  This was i n t r i g u i n g , as p u r i f i e d  at or below pH 6.5 (20).  contradictory,  of t h i s  the permeabilizing e f f e c t s of MCPs  enhanced a t acid pH ( F i g . MCPs  observation  the  killing  enzymes.  of human  different  polymorphonuclear  bactericidal  52  proteins  leukocytes have  been  i d e n t i f i e d and  characterized  (Table 1).  To date, a s i n g l e 37  protein has been found to possess optimal k i l l i n g a c t i v i t y at 5.5,  while  presence  of  the  c a t i o n i c proteins with  The  different  pH  optima would allow human PMNs to exert antimicrobial an extended amount of time a f t e r uptake (47).  Suoh a  a o t i v i t y for complement  of a n t i b a c t e r i a l proteins p o t e n t i a l l y exists i n macrophages, has yet to be  eluoidated.  53  pH  7.5.  optima f o r the other proteins i s pH  antimicrobial  kD  but  LITERATURE CITED 1.  Bader, J . and M. Teuber. 1973. Binding of the O-antigenio lipopolysacoharide of Salmonella typhimurium. Z. Naturforsoh. T e i l C. 28:422-430.  2.  Cech, P. and R.I. Lehrer. 1984. Phagolysosomal pH of human neutrophils. Blood. 63:88-95.  3.  Darveau, R.P. and R.E.W. Hancock. 1983. Procedure f o r i s o l a t i o n of b a c t e r i a l lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium s t r a i n s . J . 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