Download PHYSIOLOGICAL AND CHEMICAL CHARACTERISTICS OF

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2003, 54, 2, 283–290
www.jpp.krakow.pl
D. LAUBITZ
, R. ZABIELSKI , J. WOLIÑSKI , J. NIEMINUSZCZY , E. GRZESIUK
1,2
2
2
1
1
PHYSIOLOGICAL AND CHEMICAL CHARACTERISTICS
OF ANTIBACTERIAL ACTIVITY OF PANCREATIC JUICE
1
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland;
2
The Kielanowski Institute of Animal Physiology and Nutrition,
Polish Academy of Sciences, Jab³onna, Poland;
Attempts were made to find and characterize an antibacterial activity (ABA) factor
in porcine pancreatic juice (PJ). Its isolation requires several steps. Since ABA factor
was found to be heat resistant, the first step was heating for 30 min at 65 °C.
Afterwards column chromatography, ethanol precipitation and polyacrylamide gel
electrophoresis were involved. Finally, we obtained a pancreatic juice fraction with
antibacterial activity against Escherichia coli strain AB1157. In the presence of this
fraction the number of living bacterial cells in overnight culture decreased about
10,000 fold and a spot-test gave clearly positive results. The results of analysis
suggest that the antibacterial factor is a polypeptide active in a pH range 8.0 - 8.5,
that migrates in polyacrylamide gel electrophoresis as a band under 14,000 Da. Mass
spectroscopy
analysis
of
active
fraction
showed
high
concentration
of
porcine
pancreatic spasmolytic polypeptide (PSP). In conclusion, a polypeptide controlling
bacterial homeostasis has been found in the porcine pancreatic juice.
Key
w o r d s : antimicrobial factor; pancreatic juice; E.coli; protein purification; gut and
pancreas homeostasis.
INTRODUCTION
Multicellular organisms live in harmony with microbes. The gastrointestinal
tracts
of
vertebrates
may
be
colonized
by
large
number
of
prokaryotic
and
eukaryotic microbial cells. The regulation of intestinal microbial homeostasis is
a complex phenomenon involving electrical and motor activity, gastrointestinal
secretion of saliva, gastric and intestinal juices and bile, and specific cell-to-cell
284
interactions
(1,2).
The
maintenance
of
a
proper
quantity
and
spectrum
of
microbial species is especially important for the host; a misbalance may result in
often fatal consequences (3, 4). Since individual parts of the digestive tract differ
with regard to the number of microbial cells and composition of species (5), the
presence of an additional antimicrobial mechanism can be expected. Indeed, the
predominance
of
Gram-positive
bacteria
in
the
upper
intestine
(6)
and
the
observation that a healthy pancreas is resistant to bacterial infection (7) led
scientists to search for antimicrobial factors in pancreatic juice. Rubinstein et al.
(8) have described antibacterial activity (ABA) of a molecular weight of < 4,000
Da
from
canine
pancreatic
juice
(PJ)
that
demonstrated
an
intrinsic
activity
against several bacterial species. The antibacterial factor was dialyzable, pHdependent and heat resistant. The aim of this study was to isolate, concentrate and
purify an antimicrobial factor from pig PJ.
MATERIALS AND METHODS
Source of pancreatic juice
Animal studies have been approved by the Local Ethical Committee. Studies were performed
on pancreatic juice (PJ) obtained from conscious weaned pigs (10-25 kg of body weight, n=7)
surgically implanted with a catheter in the accessory pancreatic duct to collect pure, non-activated
PJ. A silicone cannula was implanted into the duodenum for introduction of the PJ between the
experiments (9). After one week post-surgical recovery the PJ was collected every 2 - 3 days in
freely moving animals. The collections started from the 4
th
h after the morning feeding and were
continued for 2 to 3 hours. Pooled PJ samples were immediately frozen and stored at -20 °C, for up
to a few days before analyses.
The isolation of the antibacterial factor
Before analysis the PJ was thawed and its enzymes were inactivated by incubation at 65 °C for
30 min, centrifuged (15000 g, 30 min), ethanol extracted and lyophilised. Samples were dissolved
in water to a volume 100-times less than that of original PJ sample, and passed through a Bio-Gel
P4 (Bio-Rad) 27 x 1,000 mm column with 10 mmol l
-1
Tris -HCl (Sigma) buffer pH 8.4 as the
eluent. The separated peptides ranged from 600 to 6,000 Da. Fractions of 10 ml were collected and
their OD was read at 280 nm, and electrophoresis was performed in Tricine-glycine polyacrylamide
gel at a constant current of 80 mA (10).
Assay of an antibacterial activity
Antibacterial activity (ABA) of the PJ sample was tested against Escherichia coli strain K12
AB1157 by two methods: in liquid LB medium (Luria Broth, Difco) and on LB plates (spot-test). In
the first method bacteria were grown overnight in LB liquid medium, diluted 100-fold (to ~10
7
ml ) with 10 mmol l
-1
-1
cells
Tris-HCl buffer pH 8.4 enriched with casamino acids (0.2%) and glucose
(0.5%), and subsequently divided into 2 ml portions placed in 16 ml tubes. Different PJ samples were
then added to the tubes, the suspensions were supplemented with buffer to a final volume of 3 ml and
incubated at 37 °C with shaking for 3 or 20 h. Total amount of bacteria was estimated by measuring
285
the OD600 nm, and the number of living cells (CFU) was determined. In addition, 100
µl culture samples
were stained with methylene blue for analysis under optical microscope (1200x).
For a spot-test, a 100
µl of overnight bacterial culture was added to 4 ml of LCA medium (LCA
was LB supplemented with 25 mmol l
-1
onto LB plates. A 30
CaCl2, 0.02% MgSO4 and 0.6% of Difco agar) and poured
µl samples at various stages of ABA purification were applied to the plates,
dried and incubated at 37 °C for 18 h.
Mass spectrometry analysis
Pancreatic juice sample was reduced, alkylated and digested with trypsin (sequencing grade Promega) following a standard protocol. Sample was applied to RP-18 precolumn (LC Packings)
using
the
0,1%
TFA
mobile
phase
and
than
transferred
to
nano-HPLC
RP-18
column
(LC
Packings) using an acetonitrile gradient in the presence of 0,05% formic acid with the flow rate of
200 nl min . Column outlet was directly coupled to nano-Z-spray ion source of Q-Tof electrospray
-1
mass spectrometer (Micromass) working in the regime of data dependent MS to MS/MS switch,
allowing for 3 seconds sequencing scan for each detected peptide. The data were analysed using
MassLynx software.
RESULTS
Initially the antibacterial activity of freshly collected pancreatic juice was
investigated. 0.5 ml samples of heated PJ were added to 3 ml of bacterial
suspension containing about 2 x 10
7
cells ml
-1
and incubated for 3 and 20 h at
37 °C with shaking; the control sample contained 10 mmol l
-1
Tris-HCl pH 8.4
instead of PJ. After 3 h, the control sample showed a 6.5-fold increase in the
number of living cells whereas in samples with PJ the number of bacteria
increased only twice. After 20 h of incubation the difference between control
and PJ samples was dramatic: the number of living cells in control increased
over 13-fold (to about 3 x 10
it decreased to 4 x 10
4
8
cells ml ) but in samples supplemented with PJ,
cells ml
-1
-1
(0.2 % of the initial number of bacteria).
Partially purified and 100-fold concentrated ABA factor was passed through a
Bio-Gel column. Among the total 65 fractions the substance exhibiting activity
against E. coli, as shown by the liquid medium test, was eluted in fractions 25 28 (Fig.1). Samples from each fraction tested negative by the spot-test, probably
on account of the low concentration of the antibacterial factor in the column
effluent. The antibacterial profile of the Bio-Gel P4 column effluent is shown on
Fig. 1. The presumed molecular weight of the fraction containing ABA was <
5,000 Da. This fraction reacted positively with the Bradford and Lowry reagents,
suggesting that the PJ antibacterial factor is a polypeptide.
PJ treatment by heating followed by ethanol extraction and lyophilization led
to
the
concentration
and
partial
purification
of
the
antibacterial
factor
(see
Materials and Methods). Figure 2 shows bacterial growth in the presence of both
thermally inactivated, and thermally inactivated and concentrated PJ. The results
of
polyacrylamide
gel
electrophoresis
proved
that
the
procedure
involving
286
Figure 1 Elution profile (solid line) obtained for Bio-Gel P6 filtration of porcine pancreatic juice. The
fractional range was 600 - 6.000 Da. The eluent optical density was read at a wavelength of 280 nm.
Total column volume was 510 ml, fraction size - 10 ml, flow rate - 1,5 ml min . The broken line
-1
shows the ABA profile of the column flow-through. 2 ml of E. coli culture AB1157 strain suspension
(ca 2 x 10
7
cells ml ) were incubated for 20 h at 37 °C with 500 µl of particular PJ fraction. The
-1
control sample was bacteria incubated without PJ buffer Tris-HCl pH 8,5 - "buffer line".
ethanol extraction led to the successful concentration of antibacterial factor, as
shown also by the positive results of the spot-test. A direct dependence between
antibacterial factor concentration and clarity of spots on the bacterial lawn was
observed.
A
protein
of
antimicrobial
activity
can
be
observed
on
the
polyacrylamide gel as a band migrating at 14,000 Da (Fig. 3).
Incubation of the bacteria with ABA from porcine PJ caused a decrease in the
optical density of the sample. This observation may suggest that the ABA factor
caused destabilisation of the bacterial cell structure (a similar mechanism has been
described for bacteria incubated with lysozyme). Microscopic analysis (Fig. 2)
confirmed this suggestion. The presence of the ABA factor in bacterial culture led
to cell wall disintegration and finally to cell lysis.
Mass
spectrometer
analysis
revealed
that
the
polypeptide
of
antibacterial
activity in pancreatic juice is the porcine spasmolytic polypeptide.
DISCUSSION
In the present study we have confirmed earlier findings by Pierzynowski et al.
(11)
on
the
presence
of
ABA
in
the
porcine
pancreatic
juice
and
further
characterized the molecule. Mass spectrometry analysis revealed that the ABA
287
A
B
Figure 2 Antibacterial activity measured by optical density OD600 of E. coli AB1157 culture treated
with different concentration of thermally inactivated (o) or thermally inactivated and concentrated
(n) PJ. Representative photographs of bacterial suspension preparation in liquid medium after 20 h
of incubation with 500 µl a) of thermally inactivated PJ (low concentration of ABA) and b) of
thermally inactivated and concentrated PJ (high concentration of ABA). Magnification - 1200x.
sequence
is
identical
to
a
106
amino
acid
porcine
pancreatic
spasmolytic
polypeptide (PSP) which was originally found as a contamination in commercial
insulin stocks prepared from porcine pancreatic extracts (12). PSP belongs to
trefoil peptides family and is composed of two homologues domains cross-linked
by seven disulphide bonds (13). From the circulatory system, PSP is taken up by
mucus producing cells in the gastrointestinal tract (14) and it was found to
stimulate
cell
migration
in
the
stomach
epithelium
(15),
and
inhibit
the
gastrointestinal motility and gastric acid secretion (16); however, the activity
against the bacteria have not been reported so far.
The molecular weights of ABA isolated from PJ of several sources differ.
Canine pancreatic juice ABA was estimated by Bio-Gel P4 filtration to be a
peptide of below 4,000 Da (8), while human ABA was assumed on the basis of
ultrafiltration through membrane filters (17), to be between 10,000-30,000 Da.
The broad range of ABA factor molecular weights may be the result of species
differences;
however,
the
effect
determination can not be excluded.
of
method
used
for
molecular
weight
288
Figure 3 Electrophoretic profile
of pancreatic juice on the TrisGlycine polyacrylamide gel run
with SDS-buffer. 1 - 5 µg BSA
(control);
-
heated
and
concentrated
PJ
(x100);
3
heated
-
PJ;
2
4
heated
PJ
-
and
filtrated, < 10 kDa fraction; 5 molecular
weight
standard
proteins. Antibacterial activities
of
each
pancreatic
juice
preparations were indicated on
the
spot-test
(bottom).
It
has
been done on E. coli AB1157
lawn
by
standard
method.
Sample volume was 30 µl each.
In our studies the molecular weight of porcine PJ ABA estimated by gel
filtration
was
below
5,000
Da,
whereas
polyacrylamide
gel
electrophoresis
showed a band of 14,000 Da. In gel filtration the samples were applied directly
whereas in SDS-PAGE electrophoresis they were denatured by boiling in buffer
containing SDS and 2-mercaptoethanol. Moreover, the migration of molecules in
gel filtration is caused by eluent pressure while in electrophoresis it is evoked by
the electrical field. The observed differences in migration through the two types
of gels may suggest the presence of a highly folded 3D molecular structure of the
ABA polypeptide, e.g. the presence of S-S bridges between amino acids what
corresponds to the PSP structure (13).
In previous studies ABA was tested in liquid media (8, 17). We can assume
that the ABA concentration in these studies was below detection in the spot-test.
In our preparations we showed antibacterial activity of porcine PJ in liquid
medium as well as in the spot-test. It should be noted that the spot-test of a
peptide of antibacterial activity cannot be compared with one of antibiotics as it
289
was done elsewhere else (11). This is because the relatively large molecule of the
antibacterial polypeptide unlike the relatively small molecules of antibiotics, can
not diffuse in agar and create a zone of dead cells. Under these circumstances, the
ABA was estimated by the clarity of the PJ spots loaded on the bacterial lawn. In
addition to successful spot-testing, we have been able to demonstrate a peptide
band of an intensity increased by purification, which migrated on polyacrylamide
gel at 14,000 Da (Fig. 3).
Rubinstein et al. (8) have shown that the ABA factor is highly sensitive to
changes in pH. Indeed, in our samples of pancreatic juice collected from one pig
in which acute pancreatitis was induced by obstructing pancreatic duct catheter
overnight, the ABA factor was absent and the juice pH was below 8.0 (own
unpublished data). This sensitivity to pH seems to be one of the regulators of
antibacterial activity. The concentration and composition of electrolytes as well
as the pH are regulated by a variety of ion pumps and channels that maintain
conditions maximizing the antimicrobial activity. A similar regulation system
was found in the cystic fibrosis (CF) syndrome where the homeostasis between a
local,
salt-dependent
antibacterial
activity
protein
(human
ß-defensin-1)
and
chloride ion concentration is disturbed (18, 19).
In summary, we have confirmed the presence of an antibacterial polypeptide
factor
in
the
pancreatic
juice
of
healthy
weaned
pigs,
concentrated
it
and
described its basic physical and chemical properties; moreover, we have adapted
the spot-test for fast and easy screening of ABA in PJ samples. Mass spectrum
analysis has shown similarity with the porcine spasmolytic polypeptide.
Acknowledgments: Dr D Laubitz was awarded a fellowship from the Foundation for Polish
Science in 2003.
REFERENCES
1.
Grzesiuk
E,
Laubitz
D,
Wojcik-Sikora
A,
Zabielski
R,
Pierzynowski
SG.
Influence
of
intestinal myoelectrical activity on the growth of Escherichia coli. Bioelectromagnetics 2001;
22:449-455.
2.
Konturek PC. Physiological, immunohistochemical and molecular aspects of gastric adaptation
3.
Budzynski A, Bobrzynski A, Lorens K, Konturek PC, Thor P, Konturek SJ. The influence of
to stress, aspirin and to H. pylori-derived gastrotoxins. J Physiol Pharmacol 1997; 48:3-42.
cholecystokinin on gastric myoelectrical activity in duodenal ulcer following Helicobacter
pylori eradication--an electrogastrographic study. J Physiol Pharmacol 2002; 53:171-82.
4.
Brzozowski T, Konturek PC, Kwiecien S, Konturek SJ, Pajdo R, Drozdowicz D, Ptak A, Pawlik
M,
Stachura
impairment
J,
Pawlik
associated
WW,
Hahn
EG.
with Helicobacter
Triple
eradication
pylori infection
in
therapy
counteracts
Mongolian
functional
gerbils. J
Physiol
Pharmacol 2003; 54:33-51.
5.
Jensen BB. The impact of feed additives on the microbial ecology of the gut in young pigs. J.
Anim. Feed Sci. 1998; 7:45-64.
290
6.
Gorbach SL, Nahas L, Weinstein L, Levitan R, Patterson JF. Studies of intestinal microflora. IV.
The microflora of ileostomy effluent: a unique microbial ecology. Gastroenterology 1967;
53:874-880.
7.
Williams
LF,
Byrne
JJ.
The
role
of
bacteria
in
hemorrhagic
pancreatitis.
1968;
Surgery
64:967-972.
8.
Rubinstein E, Mark Z, Haspel J, Ben-Ari G, Dreznik Z, Mirelman D, Tadmor A. Antibacterial
activity of the pancreatic fluid. Gastroenterology 1985; 88:927-932.
9.
Zabielski R, Lesniewska V, Guilloteau P. Collection of pancreatic juice in experimental animals:
mini-review of materials and methods. Reprod. Nutr. Develop. 1997; 37:385-399.
10. Schagger
H,
von
Jagow
G.
Tricine-sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem.
1987; 166:368-379.
11. Pierzynowski
postnatal
SG,
Sharma
developmental
P,
Sobczyk
stages
on
S,
Garwacki
antibacterial
S.
Influence
activity
of
the
of
feeding
pancreatic
regiment
juice.
and
Int.
J.
Pancreatol. 1992; 12:121-125.
12. Jorgensen KH, Thim L, Jacobsen HE. Pancreatic Spasmolytic Polypeptide (PSP): Preparation
and initial chemical charactreryzation of a new polypeptide from porcine pancreas. Reg. Peptid.
1982; 3:207-219.
13. Thim L. A new family of growth factor-like peptides. 'Treofil' disulphide loop structures as a
common feature in brest cancer associated peptide (pS2), pancreatic spasmolytic polypeptide
(PSP) and frog skin peptides (spasmolysins). FEBS Letters 1989; 250:55-90.
14. Poulsen
SS,
Thulesen
J,
Nexo
E,
Thim
L.
Distribution
and
metabolism
of
intravenously
administered trefoil factor 2/porcine spasmolytic polypeptide in the rat. Gut 1998; 43(2):240-7.
15. McKenzie C, Marchbank T, Playford RJ, Otto W, Thim L, Parsons ME. Pancreatic spasmolytic
polypeptide protects the gastric mucosa but does not inhibit acid secretion or motility. Am J
Physiol 1997; 273(1 Pt 1): G112-117.
16. Jorgensen
KD,
Diamant
B,
Jorgensen
KH,
Thim
L.
Pancreatic
Spasmolytic
Polypeptide
(PSP):Pharmacology of a new porcine pancreatic polypeptide with spasmolytic and gastric acid
secretion inhibitory effects. Reg. Pept. 1982; 3:231-243.
17. Bertazzoni Minelli E, Benini A, Bassi C, et al. Antimicrobial activity of human pancreatic juice
and its interaction with antibiotics. Antimicrob. Agents Chemother. 1996; 40:2099-2105.
18. Smith JJ, Travis SM, Greenberg EP, Welsh MJ. Cystic fibrosis airway epithelia fail to kill
bacteria because of abnormal airway surface fluid. Cell 1996; 85:229-236.
19. Goldman MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M, Wilson JM. Human betadefensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 1997;
88:553-560.
Received:
April 18, 2003
Accepted:
April 24, 2003
Author’s address: Dr. El¿bieta Grzesiuk, Institute of Biochemistry and Biophysics, Polish
Academy of Sciences, Pawiñskiego 5A, 02-106 Warszawa, Poland. Tel.: + 48 22 659 70 72 ext.
3337, Fax: + 48 39 12 16 23.
E-mail: [email protected]