ANTIBACTERIAL ACTIVITY OF SEMINALPLASMIN

70

3.1 INTRODUCTION

Seminalplasmin (SPLN) exhibits antimicrobial activity against both gram-positive and

gram-negative bacteria as well as yeast (Shivaji et al., 1989). In a preliminary report, SPLN has

also been shown to associate with model membranes of phosphatidylcholine and phosphatidic acid

(Galla et al.,1985), and bind strongly to sperm plasma and acrosomal membranes (Shivaji, 1986).

Analysis of the sequence of SPLN (Chapter 2) indicates the pres-ence of two putative amphiphilic

regions between residues 28-40 and 14-26. It is conceivable that the antibacterial activity of

SPLN stems from its ability to change the permeability properties of the bacterial plasma

membrane. Hence the effect of SPLN on the inner membrane permeability of bacteria was studied.

3.2 MATERIALS AND METHODS

Seminalplasmin

SPLN was prepared as described by Reddy and Bhargava (1979) and as described in Chapter 2.

Antibacterial Assays

Antibacterial activity of SPLN in the absence or presence of divalent cations was determined

by incubating logarithmically growing cultures of E. coli W 160-37 (an arginine auxotroph

derived from E. coli K-12) diluted to an 00 of 0.01 at 600 nm in the absence or presence of

various concentrations of the appropriate cations at 37oc for 6h and measuring the 00 at 600 nm.

The growth in the absence of SPLN under similar conditions was taken as control. The composition of

the synthetic medium in which the cells were grown was NH4CI, 0.5 g; (NH4)2S04, 0.5 g;

KH2P04, 13.6 g; MgS04, 7H20. 0.02 g; (NH4)2 Fe(S04)2. 6H20. 0.0156 g; glucose, 20 g and L­

arginine HCI 0.1 g per litre of the solution.

71

Bacteriolytic activity

Bacteriolytic activity of SPLN was monitored by following decrease in optical density at 600

nm (00600) of E. coli W 160-37 cells grown to logarithmic phase at 37oc in minimal A medium

(Miller, 1972) (10.5g KH2P04, 4.5g K2HP04, 1g of (NH4)2S04, O.Sg sodium citrate, 0.1 g

M g S 0 4, 0.1 g-L-arginine and 1% glucose in 1 litre of water] in the presence of 5 x 1o-4M

isopropyl thiogalactoside (IPTG). The 00 of the culture before adding SPLN was 0.2 to 0.3 (2-3 x

1 o8 cells/ml). Aliquots from the bacterial cells incubated with or without SPLN were withdrawn

at defined time periods and 00600 was measured. Bacteriolytic activity was also monitored by

measuring the release of ~-galactosidase from the cells in the presence of SPLN. Aliquots of 150 Jll

were drawn at the same time intervals and diluted to 1 ml with assay buffer (0.06M Na2HP04,

0.04M NaH2P04, 0.01 M KCI, 0.001 M MgS04 and O.OSM mercaptoethanoL, pH 7.0} and spun down

at 12000 x g at 4oc. ~-galactosidase activity in the supernatant was measu-red using

orthonitrophenylgalactoside (ONPG}. In order to determine the permeability properties of the

bacterial inner membrane in the presence of SPLN, the influx of ONPG into the sedimented cells

from above was determined by incubating the cells in 1 ml of the assay buffer with ONPG at 37oc.

The total enzyme activity of the sedimented cells in the absence of SPLN was measured after treating

the cells with a few drops of 0.1% SOS and chloroform.

Preparation of spheroplasts from E. coli cells

Spheroplasts from E. coli W 160-37 cells were prepared as described by Ito et al.,

(1977}. E. coli cells were grown in the above synthetic medium in the presence of 5 x 1 o-4 M

IPTG to an 00 of 0.4 at 600 nm. The bacterial pellet from approximately 50 ml the culture was

washed with 0.05 M Tris HCI (pH 8.1} suspended in 0.2 ml of 20% sucrose solution containing

0.03M Tris HCI (pH 8.1} and cells were converted into spheroplasts by incubation in an ice bath for

30 min with 1/10 volume of 1 mg/ml lysozyme freshly dissolved in 0.1 M EOTA (pH 7.0}.

72

Spheroplasts were collected and resuspended in 10 ml synthetic medium containing 20% sucrose.

Aliquots of 1 ml of this prepration were used for lysis experiments.

The lytic activity of SPLN on the spheroplasts was determined by measuring the release of ~­

galactosidase after incubating aliquots of spheroplasts for 20 min with varying concentrations of

SPLN at 37oc in 20% sucrose solution and determining the ~-galactosidase activity (Miller, 1972)

with ONPG in the supernatant and the pellets, after centrifugation at 15000 x g at 4oc. ~­

galactosidase released as a result of treating the spheroplasts with water was taken as 100%. For

determining the effect of divalent cations, the experiment was carried out in the presence of varying

concentrations of the appropriate cation.

Osmotic protection experiments

Spheroplasts prepared from E. coli were suspended in 20% sucrose solution in minimal A

medium and 30 mM of polyethylene glycol (PEG) of molecular mass 600, 1540, 3000 or 4000.

Then, SPLN (1 011M) was added and the ~-galactosidase activity was assayed after incubation for 20

min.

Assay for release of proteolytic activity in the medium on incubation of the culture

in the presence of cations

E. coli W 160-37 was grown in minimal A medium in the presence of 0.48 mM calcium

lactate or manganese acetate or zinc acetate to an 00600 of 0.6. The culture was filtered through a

membrane filter of 0.45 11m (millipore). It was then assayed for proteolytic activity on

carboxymethylated lysozyme and on SPLN.

For the assay on lysozyme, 170 111 of the above culture filtrate was incubated with 300 11g of

carboxymethylated lysozyme at 370C for 1 h and the mixture then run on a 11-Bondapak C1a using a

gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid (TFA) in 30 min at a flow rate of 1

ml/min. Lysozyme was identified by the eluting time and estimated by computerised evaluation of

the peak area.

73

For the assay on SPLN, 170 J.LI of the culture filtrate was incubated with 300 Jlg of SPLN for

1 h at 370C. The antibacterial activity was determined by serial two fold dilution (the lowest

dilution was 8-fold) in a micro test plate to give different concentrations of SPLN. An

appropriately diluted bacterial culture (50 Jll containing 200-500 cells) in the logarithmic phase

was added to each well. The final volume in each well was 100 Jll. The plate was incubated at 370C

and the wells scored visually for turbidity after 24h. A control,with an incubated SPLN alone, was

run concurrently. The highest dilution showing no turbidity was taken as the titre for the SPLN

sample. ca2+ at the concentration (<0.05 mM) present in the incubation medium as a. result of

carry over had no affect on the inhibition of growth of E. coli by the SPLN present in the medium.

3. 3 RESULTS

In order to ascertain the permeability status of the bacterial inner membrane in the

presence of SPLN, the activity of ~-galactosidase, a cytoplasmic enzyme was monitored. The activity

of ~-galactosidase in the cel.l-free supernatant and in the cell pellet as a function of time., in the

presence of 30 J.1M SPLN is shown in Fig. 3.1. Considerable increase Jn_ ~-galactosidase activity,

with increasing time, in the pelleted cells, is discernible upon incubation of the cells with SPLN.

However, very little ~-galactosidase is released into the cell-free supernatant. It is well

established that influx of ONPG through the bacterial inner membrane occurs through lac permease,

and in the absence of the protein transporter, no influx of ONPG is possibie. Enhanced activity of ~­

galactosidase in the presence of an antibacterial agent would reflect on the permeabilization of the

bacterial membrane to ONPG (Lehrer eta1.,1989). Hence, SPLN clearly permeabilises the bacterial

inner membrane providing additional pathways for ONPG influx. It is unlikely that SPLN gets

internalized, as there is an increase in the ~-galactosidase activity as a function of time reflecting

continued influx of ONPG. If there was internalization of SPLN, there could only be an initial

increase in the permeability.

The absence of ~-galactosidase activity in the supernatant indicates that SPLN does not lyse

bacterial cells in the time course of the experiment. During the course of the experiment,there is a

decrease in ODsoo from 0.283 to 0.119. Although this decrease is more than 50%, the amount of

>-1--> -1-(.) <t

...J

~ 0 1-~ 0

Fig. 3.1

100 0·5

, , .,.. .,. .,...,.. .,..,.. 0·4 80

.,.. .,.. .,.. .,. ~ .,. .,. .,.

~ 0 .,.. 0·3 60 ,

0 , ,, (0 ...

0 0

40 0·2

0·1

TIME (min)

Effect of SPLN on the influx of ONPG into E. coli cells. (0) influx of ONPG into

cells in the absence of SPLN, (•) influx in1o cells in the presence of 30 ~M

SPLN. 13-galactosidase activity in the supernatant in the absence of (Li) and

presence of (.&) 30 ~M SPLN. ODsoo of the cells as a function of time monitored

in the absence (--) and presence (-.-) of SPLN is also shown. Total 13-

galactosidase activity was determined by treating the cells with a few drops of

0.1% SDS/chloroform. The values on the ordinate are percentage of the total

activity normalised to ODsoo of 1 and are taken as an indication of influx of

75

~-galactosidase detected in the supernantant is not substantial. SPLN did not inhibit the activity of

purified ~-galactosidase, indicating that lack of activity in the supernatant is not due to inhibition

of the activity by SPLN.

SPLN has been shown to lyse bacteria at higher concentrations than required for inhibition of

growth by activating the autolysins (Chitnis et al., 1987). It has also been shown that E. coli cells

grown at pH 5.0, where autolysins are not activated (Goodell et al., 1976) were much less

susceptible to lysis by SPLN. In order to understand whether or not autolysins were involved in

bringing about the inner membrane permeability changes in E. coli, the influx of ONPG with E. coli

cells in the presence of SPLN under this condition was investigated. Increase in ONPG influx into E.

coli cells as a function of time in the presence of SPLN was observed even under this condition {Fig.

3 .. 2)

It has been well established that divalent cations like ca2+ play an important role in

stabilising the structure of membranes, particularly the outer membrane of gram-negative

bacteria {Lugtenberg and van Alphen, t983; Nikaido and Vaara, 1985). Hence the effect of these

cations on the antibacterial activity and membrane permeabilising activity of SPLN were examined.

ca2+, Mn2+ and zn2+ when added with SPLN clearly reverse the inhibition of growth of E. coli by

the above protein to an extent of 70 to 92% {Table 3.1 ). This is most probably brought about by

stabilising the outer membrar:~e structure and preventing the entry of SPLN into bacterial cells and

localizing in the inner membrane.

The reversal of inhibition of growth of E. co1i by cations could also arise due to the release

of a proteolytic enzyme by the bacteria in the presence of the cations. Hence the culture-filtrate

obtained following the growth of E. coli in the presence of 0.48mM calcium lactate, or manganese

acetate, or zinc acetate was tested for the presence of proteolytic activity on lysozyme {Fig. 3.3) and

SPLN {Table 3.2) as described in Materials and Methods. There was no evidence whatsoever of any

degradation of the two proteins by this culture filtrate.

In order to get further insight into the nature of the pathway for ONPG in the inner

membrane as a result of perturbation by SPLN, experiments were performed by using the

76

(!) a_ z 0 lL. 0

X :::) _j

lL. z ~ 0 20

0 100

TIME (min)

Fig. 3.2 ONPG influx into E. coli cells grown at pH 5.0. (0) Influx in the absence of

SPLN, (0) Influx in the presence of SPLN (30 11M).

77

Table 3.1

Effect of divalent cations on the inhibition of growth of E. coli W160-37 by

SPLN

(~g/ml)

0

3 0

0

30

3 0

3 0

0

30

30

0

30

3 0

Cation Cone

(mM)

None

N-one

zn++ 0.72

zn+ + 0.24

zn+ + 0.4"8

Zri+ + 0.72

ca++ 0.72

ea++ 0.24

ca+-+ 0.48

Mn-++ 0.72

M=n+ + 0.24

Mn+ + 0.48

seminalplasmin

A760

of the

culture

at 6h

0.5

0.0

0.53

0.0$

0.12

0.35

0.57

0.24

0.42

0.50

0.13

0.46

Inhibition

of growth

(2)

100

0

82

76

30

G

78

27

G

74

8

Relief of in­

hibition by

the

cation (%)

1 8

2 4

70

2 2

73

-.

2 6

9 2

---------------------------------------------------

100 (1

80

- 60 ---- 40 ~ 0

- l>

- 20 n - rT1 ~ -- -i l() - 0 0 C\1 100

<:t b z -i

80 ::0 r rn

- 60 ----- 40 -20 --- .. L __

0 4 8 16 20 24 ~8 32 0

36 40

TIME (min)

Fig. 3.3 Proteolytic activity of cell tree culture filtrate from E. coli. Cells grown in the

presence of ca2+ on carboxymelhylated lysozyme. HPLC profiles of

carboxymethyiated iysozyme (a) unincubated and {b) "-1

incubated with the cell free culture-filtrate. (Details are in text). 00

79

Table 3.2

Effect of incubation of seminalplasmin with cell free culture-filtrate of E. coli

grown in the presence of divalent cations on its antibacterial activity.

SPLN incubated with MIC (Jl g I m I)

1 . S-ynthetic medium 50

2. Syn th·eti-c med h:rm 50

C:Onta:ining Ca2 +

3. -Filtered med i:um f.rom E. coli 50

cells grow.n in the synthe.Uc

medium conta-ini-ng ca2 +

Seminaf:pJasmin after CM-Sephadex stage which was about 60% pure was used fo-r

this experiment and in the control 50 Jlg/ml seminalplasmin was required for

10.0% growth inhibition

80

spheroplasts made from E. coli. Unlike in the case of intact cells, ~-galactosidase activity could be

detected in the supernatant after spinning down the spheroplasts. Fig. 3.4. shows the release of ~­

galactosidase as a function of SPLN concentration. It is ev-ident that ~-galactosidase is released even

at very low concentrations of SPLN, indicating that the inner membrane is more susceptible to

perturbation by SPLN than the outer membrane.

In order to determine whether the lysis of the spheroplasts proceeds through a colloid

osmotic mechanism, and also to determine the size of the lesion produced, release of ~-galactosidase

in the presence of various osmoprotectants was determined. Table 3.3 shows the results of this

• study. It is evident that about 40% protection to lysis is obtained in the presence pf PEG 4.0{)0,

0

indicating the presence of lesions of - 40 A (Scherrer and Gerhardt, 1971 ). Thus, lysis JilrGCeeds

through a col~o~d osmotic mechanism.

As divaient cations :(Drovide protection against the growth-inhibitory effect o-f SPLN on

bacteria, the effect of these ca1ions en the leakage of ~-galactosidase from the spheroplasts ln the

pr:esence of SPLN wa-s examio.e.c:L Protection to lysis is not obtained at m-icromolar levels o-f the

cations) as obtained in the case of growth inhibition (Table- 3.4). However1 protection is se'en in t-he

millimolar ranges of ca2+. Extensive studies on the membrane .damag-e induced by dilferer:rt

hemo.lytic ag-ents, by measuring the leakag-e of monovalent -cat-io-r:~s and hemolysis o-f erythr-o-cyt-es,

have indicated that div~lent cations like c.a2+ protect cells against m-embrane damage a-t the

extraceJlular side of the plasma m-embrane. Such a protecnon is obtained at millimolar lev-els o-f

cations, presumably by ch-elating the negative.ly charged groups of the pores on the external side of

plasma membrane (Bash.f.ord etaL, 1986). Thus, the lysis of spheroplasts by SPLN pr:oceeds

through a colloid osmotic mechanism, similar to the lysis of red blood cells by various hemoiytic

a§ents.

3.4 DISCUSS10N

SPLN has a potent antimicrobial activity. The effect of SPLN on the macromolecular

synthesis (DNA, RNA and proteins) in whole cells of E. coli (Reddy and Bhargava, 1979), C.

:a1bicans (Scheit and Bhargava, 1985) and S. cerevisiae (Scheit et al., 1985) has been studied. It

81

0 w

100 (/) <{ lJ.J _J w

80 ·~

w (/) <{

60 0 (/)

0 ...._ (.) 40 <{ _j <{ <..!)

·~ 201 .· 0 l(_ 0'

_ _L

0 2 4 6 8 LO

SPLN ()J M)

Fig. 3.4 Release of ~-galactosidase from E. coli spheroplasts in the presence of SPLN.

82

Table 3.3

Protection to lysis of sph-eroplasts provided by various osmoprotectants (30 mM)

in 1he presence of 10 J.LM SPLN

Protectant

None

PEG GOO

PEG 1540

PEG 3000

PEG 4000

% ~-gal released % Protection

76

74

58 24

55 28

50 38

83

Table 3.4

Protection to spheroplast-lysis by cations

Spheroplast- SPLN CaCI2 00420 Lysis Protection

suspension cone cone %

J1M

-------------------------------------------------------------

1 m'l 0 0. 0 8 4

1 mt 10 0.270 1 0 0

1 ml 10 200J.tM 0. 2 6 3 1 0 0 0

1 ml 10 400J.tM 0. 2 63 1 0 0 0

1 ml 10 600J1M 0. 2 6 2 1 0 0 0

1 mJ 10 15mM 0.1 9 6 56 44

1 ml 10 3:QmM 0.1 2 7 22 78

1 ml 10 45mM 0. 0 4 3 0 100

84

has been found that in all the cells RNA synthesis is inhibited strongly except in the case of isolated

mitochondria, where the inhibition is weak (Shivaji et at., 198"9). DNA and protein synthesis are

not affected or are inhibited much more weakly than RNA synthesis. It is believed that the inhibition

of RNA synthesis is a primary effect of SPLN that leads to the inhibition of growth of

microorganisms, even though there is no direct demonst-ration of the entry of SPLN into the

cytoplasm to bring about this activity. As it was also known that SPLN interacts strongly with

membranes (Galla et at., 1985; Shivaji,1986), the effect of SPLN on the membrane permeability

of E. coli was studied. The results show that SPLN has the ability to alter the permeability

properties of the bacterial membrane as judged by the increased ONPG influx as a function of time.

Decrease in the efflux of ~-galactosidase from spheroplasts in the presence of osmoprotectants and

millimolar concentrations of divalent cations indicates that a colloid o-smotic type of lysis

mechanism operates. Since the influx of ONPG into E. coli cells increases with time at a fixed

peptide concentration, it is u:n:likeJy that SPLN is interna4iz,erl and exerts its activity on RNA

synthesis machinery. The inner membrane of bacteria contains the electron transport chain and

enzymatic system-s for ene:rgy generation. It is likely that associat-ion of SPLN r-es:ults in extensive

uncot:~pling of various pumps and other translocators. This ca-uses gerre-ralis-es permeability

breakdown ar:~d subsequently cell death. Although autoly:sins are b_elieved to play a role in the

antibacterial activ.ity of S:PLN (Chitnis et at., 1987) as .they are known to be induced due to

perturbation of inner membrane (Goessens et at., 1988]), it is unlik-ely that h;u;!uction o1 auto1ysins

plays a majo.r role in the antibacterial activity of SPLN, as increased ONPG intlu:x is also observed at

pH 5.0_, when autolysin induction does not take place (Goodell et at., 1976).

The cell envelopes of gram-negative bacteria (Fig 3.5) such as E. coli are complex in terms

of chemistry and structure (Lugtenberg and van Alphen,1983; Storm et al.,1977). They consist of

an external outer membrane that overlies a thin layer of peptidoglycan. Freeze substitution

preserves th-e periplasm efficiently and reveals a 'periplasmic gel' between the outer and inner

membranes. The outer membrane consists of proteins, phospholipids and lipopolysaccharides

arranged in an exact format. Almost all the lipopolysaccharide is appropriated to the external face

F1g. 3.5

CAPSULE

OUTER MEMBRANE

P£-IU.PLASM

PEPT L60·GL¥CAN

!NNE R MfM'BRA N:E

The cell envelope of a gram negative bacterium. LP

li-pop-rotein: LPS

ph o·s p-h ai ip+d .

lipepolysaccharide: P - protein: PL

85

86

of the membrane, whereas the phospholipid aligns along the inner face. Proteins are distributed

throughout the membrane and can be oriented on either surface or can span it completely.

The outer and inner membranes of gram-negative bacteria present barriers to the entry of

many compounds into the cytoplasm (Hancock, 1984). Small hydrophilic molecules are able to

cross the outer membrane either by passive diffusion· through nonspecific diffusion pores such as

those formed by the omp F or ompC porins, or by transport through specific different channels such

as th?se composed of the Lam B protein which facilitates the transport of maltodextrins across the

outer membrane (Nikaido and Vaara, 1985). In E. coli porin consists of two chemical varieties,

whose levels are modulated by the osmolarity of the external milieu. The omp F polypeptide is

slightly larger and more basic than omp C polypeptide, but each has the same function within the

outer membrane. They are intrinsic proteins that are closely associated wHh Upoprotein and

lipopolysaccharide and form small, hydrophilic channels that span the bilayer. Hydrophilic

molecules with molecular weight of about 600-1000 ca.n percolate througn ttlese channels, but

larger molecules are excluded. It is thus a sieving mechanism. SPLN, with a molecular weight

>50ou·, canBoJ cross these channels for entry into bacterial cells for its locaHza1ion in the inner

membrane.

Macromolecules such as colicins or the genome-5 of certain bacte.riophages have taken

advantage O;f other systems iB E. coli for entry into the membrane or cyteplasm. C.olicins or

bacteriocins, are antagonist~c to 9acteria not capable of producing them (Konlsky, 1982). Their

target of action varies, ranging from cleavage activity on DNA (Colicin E2) or RNA (Colicin E3) to

lysis of bacteria (Colicin M) or the ability to form ion permeable channels in the membrane of

susceptible bacteria (Colicin A, B, E, V, W, 1 a, 1 b). In general, the transport of these molecules to

their targets requires interaction with high affinity receptors in the outer membrane (Webster,

1991 ). As SPLN acts on a variety of microorganisms including gram-positive bacteria and other

cells nonspecifically, it is unlik~ly that SPLN uses these receptors for entry into cells. This

argument is strengthened by the fact that divalent cations at micromolar concentrations inhibit the

antibacterial activity of SPLN.

87

Since SPLN would be cationic at pH 7.4, it" can associate with the outer membrane, like

polymyxin and polylysine and perturb the outer membrane to an extent that it can gain entry to the

inner membrane. Thus, the decrease in OD shown in Fig. 3.1. does not appear to signify cell lysis

and probably reflects disruption of the outer membrane. Even though reduction of turbidity is in

most cases attributed to cell lysis and loss of viability, it has been shown that reduction in turbidity

need not necessarily be associated with reduction in cell number (McDowell and Reed, 1989).

Divalent cations and particularly Ca2+ have been shown to be responsible for the

stabilization of the lipopolysaccharide in the lipid domain of the outer membrane (Lugtenberg and

van Alphen, 1983; N4kaido and Vaara, 1985). Presumably, Ca2+ bo.und to the outer membrane

reduces charge repulsion between ·highly anionic constituent molecules, bridges adjacent molecuJes

o-f lipopolysaccharide and (or) proteins and help anchor the outer membrane to the underlying

fabr,ic of peptidog-lycan. This by:pothesis has been supported by da1a that EDTA-modified outer

membrane o-f E. coli is Rot only deficient in lipopolysaccharides hut also in Ca2+ (Ferris and

Beveridge, r986). EDTA-tr:eated cells are typically more susceptible to b}'.dr:o!yHc enzymes and

antibiotics that are normally excluded by the outer membrane. The loss of over 40% of the outer

membrane bound-calcium during EDTA treatment increases the electrostatic repulsion between

anionic consntuents of the outer membrane and the conformational change that results in the release

oJ Hpopo:lysaccharide kom the o-uter membrane can be readily visualized as vesicular structur.-es

with a high degree of surface curvature. Divalent cations are known to antagonise several e-ffects of

polymyxin and other peptide antibiotics by preventing their entry as a result of stabilization o-f the

outer membrane (vafl Alphen, 1983; Nikaido and Vara, 1985 ). Hence, it is likely that SPLN is

prevented from reaching the inner membrane when the outer membrane is stabilised by divalent

cations.

One of the better studied antimicrobial peptides are defensins (Lehrer et al., 1991 ).

Defensins account for - 30% of the total proteins in human cytoplasmic azurophil granules of the

polymorphonuclear neutrophils (PMNs). These PMNs are the most abundant phagocytic cells

circulating in human blood and have potent oxidative and non-oxidative microbicidal mechanisms.

88

The non-oxidative mechanism are mediated by antimicrobial peptides that include defensins.

Defensins are invariably cationic , relatively arginine rich, nonglycosylated peptides composed of

39-34 residues. To date, sequences of 15 defensins from mammalian phagocytes have been

established (Fig. 3.6). All defensins contain a characteristic Cys motif, with three intramolecular

disulfide bridges. Defensins show in vitro activity against gram-positive and gram-negative

bacteria, fungi, mammalian cells and envelope viruses. The work of Lehrer and Colleagues has

shown that the defensins permeabiUse both the inner and outer membranes of E. coli and that the

inner membrane permeabilisation is coincident with cell death. HNP-1 (human neutrophil peptide

defensin-1) sequentially permeabilises the outer membrane and the inner membrane of E. coli

(Lehrer et al., 1989). Outer membrane permeabilisation could be partially dissociated from inner

membrane permeabilisation with mannitol plasmolyzed cells and under such conditions bacterial

death more closely parallels loss of inner membrane integri-ty. Thus, inner membrane

permeabilisation is the letha1 ev·ent.

Determination of the crystal structure of defens-in HNP-3 (Hill etal.,1991) has enabled

further understanding of its mechanism of action. The structure is remarkably different from other

lytic or membrane permeabilizing peptides like melittin, cecropin, magainins, alamethicin and a­

hemolysin (Eisenberg and Wessor1, 1990). All of these pe:ptides are amphiphilic a-helices that

ceRtain no ~-sheet and have no clisulfide bonds. l.n sharp .contr..ast, deJensin is an all ~-sheet protein

with no a-helix and it is stabilized by three disumd.e b:0lids..

Defensin is an elongated, ellipsoidal molecule (Hill et at, 1991) (Fig. 3.7). The structure

is quite rigid and contains a three stranded antiparaiJel ~-sheet that includes 60% of the residues.

The constraints of the three disulfide bonds prohibit a gross conformational change, upon moving

from an aqueous to an apolar environment. Thus, the crystal structure promotes a good model for

the conformation in solution and at the site of action. The pattern of conserved residues of the

defensin family suggests that all other defensins. have conformation similar to that of HNP-3. The

six Cys residues that must play a major role in stabilizing the conformation are invariant and

several other key structural residues Arg6, Glu 14 and Gly24 are found in most of the defensins.

89

r l J_ J. ,.--. r- l .r KNr-t " c J c Ill I r A c r A c I[ .... ; .. y c T c I ' 0 c Ill L N A :r c c

H'•M&a 'HNr·l HNr·l D HNr-4 v

c J c " I p A c I A c I[ " ~ y

c T '(;· I y 0' !C. k_l. w A r c c c y c " l p A c l., A Cell: ""'. J c T C '( T 0' c lll'L N A r .c c c I c " L v r c " " T ·a

L " v c tc 1 c L l c· c. v J r T :'- " "'

y

G-alua G,P'Np " " c I c ,. T " T c " r , .. , "' " L.C T!C I r 0 N " v ' T r c<:

·r1c Nl'·l v v c A c ... " A L c L p ... ;_~ .... '" :A. 0 r c. " I 1'·· :C. " J ll p .L. .c C. Ill " :-;.P·l v v C' A c " Ill A L c L p L.& " " A :C r c " I "·' ;a; Ill I • p L--C c "- " Rab1fM Nl"·l- c I NP..Jit c Ill

c A c " " " ,. c ,. .. J .c ... ,. ' c- T c " v Jl :C' A " T v " c c s " c v c " I( OLL c s T -"-~ ,& "' " t 1-a 0 _c It 1

" G v "

,. -p r c c .. " 'Ill

NP'-<4 v • Nr-s v r c T c ~ Ill ,.

' c c r .c I " A 1 ·c I c T v .. -0 v ,_ ·JC- T L- C c " II.

c T c a 0 ,. L c c I c ., -~~~- A .. c I c T l :M" ·c v " .. T L c c " " R.al'lP· J v T c ' c ~ " T " c c r " I[

~~~- L :1. C A ·C 0 J "' :C: :f'· I J -" L c c " Ral RalNP·l v T

RatNr-J RalNr-4 A

c T -c " s T " c c r Ill, I: " L I •C A c .C J 'lite '4. " I ' .... .L c c " c J c " T s s c " r c ' I' L I C A c " I. .. c .. " l ' Ill L c c c ' c " r c A c v I c --.:-

'"' ·L T c A c c L M. -C :1'1 l T .. L < c ....

~ - ~ :.____... '- - - -

Fig. 3.6 The amino acid sequen f d t · [ ces o e ens1ns from HiLl et a-1.)(1991 )].

90

0

'17 .._,,.I

91

Gly24 occupies the third position of a type I' turn; Arg6 forms a salt bridge with Gin 14 that spans

the only stretch of polypeptide that does not participate in the ~-sheet.

HNP-3 has been shown to crystallizse as a dimer and the shape of this dimer resembling a

basket with an apolar base and a polar top that includes the two amino termini and the two carboxy

termini. The ~-sheet, both twists and coils and as a result the amino terminaJ ~-strands of the two

neighbouring monomers are close together in space. Then two ~-strands are hydrogen bonded

together through ordered solvent molecules that form a mini channel, which passes right through

the dimer. The core of the basket is hydrophobic. The six Arg residues form an equatorial ring

around th'e dimer, with their side chains being relatively Hexible.

Hill e1 at, (1991) suggest t-h-ree ways in which defensins molee'tde'S might interact and

pe:rrrteabWse a 11:pid bUayer. A wedge effect might res-ult from the amphipilWci:ty of file dimer

basket. A cluster of four hydrophobic residues at the bottom of the basket prov~de-s a hydmphobic

patch of 34 A ot solvent accessible surface area that is surr:oun-cl-ed, tu·rther ap the basket, by tf:re

ring of six Arg side chains{;Rg 3.8 A & B~). ~n the wedge hyJ:>ot-hesis, the HNP-3, dim-er disrupts ttl'e

membrane by qisrupting Jip_id-lip:id interactions. The dimer does this by burying the hydrep-hobi£

s-~:~rface into the bilayer while the arginine side chains interact with the lipid phosphat-e groups.

Two ot!ler p_oss:ib:i:e m:echanisms emerge fmm th-e stwcture, both o:f whJch involv-e por-:e

fermat-ion. Or:~e o,f u~es-e, the dimer pore, makes ~;~se of the soJ~-ent mini cf:l:aRoo:l-~n in the cry;s:tal

strucrur-e. In ttris mode:l, th-e two dimers assemble in the membrane with tl:!e'ir polar tops towards

eactl ottJe;r and apolar 9ases facing Upid-taits (Fig 3.8-G). There is som-e charge complementari:t¥ a1

the putative dimer-dimer interfac-e especial-ly involving the ion pair Arg6 and Glu 14. The siEle

chains of the six "equatorial" A-f§ fesidues move to bind lipid head groups. In this conformation, two

of the solvent mini ch-annels seen in rhe crystal structure completely span th·e bilayer.

The other general pore hypothesis has dimers completely spanning the m-embrane, but now

rotated by - goo from the "dimer pore" orientation and with the polar top surface lining the pore

(Fig. 3.80). The same hydrophobic dimer surface contacts the lipid tails and Arg side chains move

ag-ains;t the l'lead groups.

A

Fig. 3.8 (A) Schematic representation of the dimer basket. The top is hydrophillc

and the base is hydrophobic :(shaded) (B) Wedge hypothesis, (C) Dtmer

pore hypothesis, (D) General pore hypothesis (details described in text)

(from Hill et a!., 1991).

93

Conformational studies have indicated that SPLN adopts an a-heHcal structure (Chapter 2)

and does not have any similarity in sequence to defensins. Thus SPLN and defensins do not share a

common structural motif. Analysis of the sequence of SPLN rev-eals at least two helical regions that

are amphiphilic and plot in the surface seeking region of the hydrophobic moment plot (Chapter 2).

The membrane permeabilising property of SPLN is likely to stem from the interaction of the two

putative amphiphilic a-helical segments with membranes. In order to examine the importance of

these segments, they were synthsized and their biological properties investigated. These are

described in subsequent chapters of this thesis.