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In vitro inhibition of adhesion of Escherichia coli
Arthur Ouwehand
Department of General and Marine Microbiology
Göteborg University
Göteborg Sweden
1
In vitro inhibition of adhesion of Escherichia coli
In vitro inhibition of adhesion of Escherichia coli.
Arthur C. Ouwehand
2
Department of General and Marine Microbiology
Göteborg University
Göteborg Sweden
3
Arthur C. Ouwehand
4
In vitro inhibition of adhesion of Escherichia coli
1996
ISBN 91-628-2194-6
5
In vitro inhibition of adhesion of Escherichia coli.
Arthur C. Ouwehand
Akademisk avhandling.
för filosofie doktorsexamen i mikrobiologi (examinator Professor Lennart
Adler), som enligt biologiska sektionsstyrelsens beslut kommer att offentlig
försvaras den 1 november 1996, kl. 10.00 i föreläsningssal A1024 Ivan
Ivarsson, Medicinaregatan 3B, Göteborg.
Göteborg 1996
ISBN 91-628-2194-6
6
Voor mam en pap
7
In vitro inhibition of adhesion of Escherichia coli
Arthur C. Ouwehand
Department of General and Marine Microbiology, Göteborg University
S-413 90 Göteborg, Sweden.
Abstract. For many Escherichia coli strains, adhesion to tissues, is
considered a prerequisite for pathogenesis. Inhibition of this adhesion, may
therefore prevent the establishment of disease in an early stage. The aim of
this work was to identify substances that may inhibit the in vitro binding to
intestinal mucus of pathogenic E. coli strains expressing either K88 or SfaII
fimbriae. Lactobacillus fermentum strain 104r had been shown previously
to produce a substance inhibitory to K88 mediated adhesion. The active
component and the mechanism of inhibition have been investigated.
Because human milk contains substances that inhibit pathogen adhesion
and antimicrobial substances have been detected in bovine colostrum, the
capacity of bovine colostrum constituents to inhibit SfaII-fimbriaemediated adhesion was investigated.
L. fermentum 104r spent culture liquid and bovine colostrum were
subjected to size exclusion chromatography and ultrafiltration followed by
anion exchange chromatography. The fractions showing adhesion
inhibitory activity were treated with different enzymes and chemicals in
order to identify the active components and determine their modes of
action. A range of Lactobacillus strains were tested for their ability to
produce similar adhesion inhibitory activity. Active components, from
Lactobacillus spent culture liquid and bovine colostrum, were examined for
activity against other enteropathogens.
It was found that L. fermentum 104r spent culture liquid contained a 1700
K carbohydrate that mediated the observed inhibition of K88 fimbrial
adhesion. The carbohydrate was concluded to be a cell wall fragment, since
the activity was affected by lysozyme treatment. No direct evidence could
be found for the binding of the carbohydrate to the intestinal mucus.
Indirect evidence suggests blocking of K88 receptors by steric hindrance,
however, the precise mode of action still remains to be elucidated. It was
observed that other Lactobacillus strains of intestinal origin also produced
inhibitory components and that the greatest inhibition was noted for
different K88 variants and SfaII fimbriae. Interestingly, the L. fermentum
104r spent culture liquid contained a high molecular weight substance
capable of enhancing the antimicrobial activity of organic acids against E.
coli growth in vitro. Its identity remains however to be determined.
It was demonstrated that bovine colostrum contained a substance that
inhibited SfaII fimbriae mediated adhesion with some effect on SfaI
fimbrial adhesion. The substance was identified as -lactoglobulin, a major
milk whey protein. -lactoglobulin binds in a concentration dependent
8
manner to intestinal mucus; using multiple receptors, and blocks, as
proposed for Lactobacillus spent culture liquid, adhesion sites by steric
hindrance. The activity of -lactoglobulin was found to be heat stable and
dependant upon the presence of 3 disulfide bridges. In all cases, the in vivo
importance of these findings remains to be determined.
It was concluded that bovine colostrum and Lactobacillus spent culture
fluid contain components inhibitory to adhesion on K88 and SfaII fimbriae.
The role of this inhibition in pathogenicity is addressed in this thesis.
Key words: Lactobacillus; Cell wall; Bovine colostrum; -lactoglobulin; Fimbriae;
ETEC
Göteborg 1996
ISBN 91-628-2194-6
9
In vitro inhibition of adhesion of Escherichia coli
by
Arthur C. Ouwehand
Department of General and Marine Microbiology, Göteborg University
Medicinaregatan 9C, S-413 90 Göteborg, Sweden
This thesis is based on the following papers, which are referred to in the
text by their respective Roman numerals:
I
Rojas, M., A.C. Ouwehand and P.L. Conway. Interactions between
high and low molecular weight compound(s) in Lactobacillus spent
culture fluid with antagonistic against E. coli K88 growth. Submitted
II
Ouwehand, A.C. and P.L. Conway. 1996. Purification and
characterization of
a component produced by Lactobacillus
fermentum that inhibits the adhesion of K88 expressing Escherichia
coli to porcine ileal mucus. J. Appl. Bact. 80:311-318.
III
Ouwehand, A.C. and P.L. Conway. 1996. Specificity of spent culture
fluids of Lactobacillus sp. to inhibit adhesion of enteropathogenic
Escherichia coli cells. Microb. Ecol. Health Dis. in press
IV
Ouwehand, A.C., P.L. Conway and S.J. Salminen. 1995. Inhibition
of S-fimbria-mediated adhesion to human ileostomy glycoproteins by
a protein isolated from bovine colostrum. Infect. Immun. 63:49174920.
V
Ouwehand, A.C., S.J. Salminen, M. Skurnik and P.L. Conway. 1996.
Inhibition of enteropathogen adhesion by -lactoglobulin. Submitted
to Infect. Immun.
10
Contents
Page
1 Introduction
1.1 General
1.2 Aims
1
1
1
2 The digestive system
2.1 Anatomy and morphology
2.1.1 The mouth
2.1.2 The oesophagus
2.1.3 The stomach
2.1.4 The small intestine
2.1.5 The large intestine, colon and caecum
2.2 Mucus
2.2.1 Function
2.2.2 Composition
2.3 The normal microflora
2.3.1 The mouth
2.3.2 The oesophagus
2.3.3 The stomach
2.3.4 The small intestine
2.3.5 The large intestine
2.4 Enteric pathogens
2.4.1 General
2.4.2 E. coli
3
3
3
3
3
5
6
6
6
7
8
8
9
9
10
10
11
11
11
3 Milk
3.1 Composition and functions
3.2 Milk whey
3.2.1 Oligosaccharides
3.2.2 Proteins
13
13
13
13
14
4 Lactobacilli17
4.1 General background
4.2 Proposed functions in the intestine
4.2.1 Colonisation resistance
17
17
18
5 Adhesion
5.1 General background
5.2 Non-specific adhesion
5.3 Specific adhesion
21
21
21
23
11
5.4 Adhesion assays
25
6 Inhibition of adhesion
6.1 General background
6.2 Affecting the adhesin
6.2.1 Blocking the adhesin
6.2.2 Reducing adhesin expression
6.3 Affecting the receptor
6.3.1 No receptor
6.3.2 Modifying the receptor
6.3.3 Specific blocking of the receptor
6.3.4 Colonisation resistance. Non-specific blocking
of the receptor
6.3.5 Lactobacillus cell wall fragments. Non-specific
blocking of the receptor
6.3.6 -lactoglobulin. Non-specific blocking of the receptor
27
27
27
27
29
30
30
30
31
7 Conclusions
40
8 Acknowledgements
42
9 References
43
31
33
37
1 Introduction
1.1 General
Many pathogens basically cause only one type of disease. Escherichia coli
strains, however, are able to cause a variety of different diseases; diarrhoea,
urinary tract infection, sepsis, meningitis etc. In order to cause disease,
pathogens possess so-called virulence factors. These include special
structures that facilitate attachment to host tissues, enzymes and other
proteins that allow invasion and translocaton, production of toxins,
capsules etc. 159. One of the first steps in the development of disease is
adhesion of the pathogen to host tissue 9, 59, 93, 152. This is especially
important in an environment as the small intestine. Once a pathogen has
adhered, it can start producing toxins; which can induce diarrhoea, or it can
invade and even translocate and cause sepsis and infect other organs.
The newborn, receives many protective factors from the milk, among them
antibodies. These passively protect the newborn. Upon weaning, this
protection seizes. When this weaning is abrupt and early, and accompanied
with other forms of stress, as in commercial pig raising, it leaves the young
12
animal very susceptible to pathogens. One of the pathogens commonly
causing disease in piglets is K88 fimbriae bearing E. coli 98. Large scale
use of antibiotics is undesirable and vaccination against post-weaning
diarrhoea is still not effective. Other ways of controling enterotoxigenic E.
coli are therefore of interest. Inhibition of adhesion may be an alternative
strategy.
Extraintestinal E. coli strains are able to cause severe sepsis and newborn
meningitis 82. Over 30% of all cases of sepsis have been found to be
caused by E. coli 64. E. coli expressing SfaII fimbriae are a major cause
of newborn meningitis and sepsis 81. Of the investigated strains isolated
from newborn meningitis, 30% produced SfaII fimbriae 104. Here too,
inhibiton of adhesion might provide a way of preventing infection at an
early stage.
1.2 Aims
The aim of this thesis was to investigate factors that might interfere with
the initial binding of K88 fimbriae and SfaII fimbriae expressing E. coli.
Component(s) produced by Lactobacillus fermentum strain 104r had
previously been found to inhibit K88 mediated adhesion 14. The aim was
to purify and characterise the component(s), investigate the mode of action
and characterise its spectrum of activity in vitro.
The gastrointestinal tract and the oropharynx have been shown to be the
main reservoir for E. coli that are able to translocate 80. Human milk has
been found to contain substances that can inhibit the adhesion of SfaII
fimbriated E. coli. Since bovine colostrum is a rich source of growth
factors, antimicrobials etc. It might also contain similar adhesion inhibitory
substances. These substances might inhibit the colonisation of these organs,
thereby reducing the risk of translocation, from the digestive tract to the
blood, and disease.
13
2 The digestive system
2.1 Anatomy, morphology and physiology.
The main function of the digestive system is the digestion of food (mouth
and especially in the stomach and the small intestine) 188, absorption of
nutrients and water (small and large intestine) 192 and the storage of
wastes (large intestine) 188. The anatomy, morphology and physiology of
the gastrointestinal tract exert a large influence on the normal microflora
and possible enteropathogens that live in the intestine. In addition,
colonisation of the intestine by the pathogens will be influenced by these
factors.
2.1.1 The Mouth
In the mouth, food is mechanically broken down into small fragments by
mastication and mixed with saliva. Saliva contains mainly water. The major
electrolytes are Na+, K+, Cl- and HCO3-, the latter functions as a buffer.
Mucus is one of the main organic constituents of saliva, it lubricates and
protects the mucosal surfaces. Digestive enzymes in saliva are -amylase
which hydrolyses -1,4 glucosidic linkages, and lipase which especially in
the neonate is important for the hydrolysis of triglycerides. Other enzymes
found in the mouth include lactoperoxidase which scavenges H2O2 (its
activity will be discussed in section 3.2.2e), and lysozyme 163 which can
act as an antimicrobial substance by hydrolysing the -1,4 bond between
N-acetylglucosamine and N-acetylmuramate in the bacterial cell wall
184. In addition to smooth and rough squamous epithelium, the mouth
also contains hydroxyapatite on the tooth surfaces to which bacteria can
adhere 188.
2.1.2 The oesophagus
The oesophagus is a hollow tube that transports the food from the mouth to
the stomach with waves of contraction passing along its length. The
peristaltic wave lasts for 7-10 seconds, but due to gravity, food may
descend more rapidly 163. The oesophagus is lined with keratinized
squamous epithelium, and may contain sparsely distributed mucus
producing cells 78.
14
2.1.3 The stomach
The stomach is a curved organ which receives ingested food and can store
it temporarily. In humans it can accommodate up to 5 liters. It functions as
a mixing and digestive organ. In the stomach, the ingested food is
transformed into a fairly homogenous product, in terms of pH, osmolality,
consistency and temperature. The central, and major, part of the stomach is
called the body or corpus. The region where the oesophagus enters the
stomach is called the cardia. In pigs, this area is called the pars oesophagus
98. Next to the cardia/pars oesophagus, is the upper most region of the
stomach, namely the fundus. The distal part of the stomach, the antrum,
merges into the pyloric canal, ending at the pyloric sphincter 188.
The pars oesophagus is covered with the same type of squamous
epithelium as the oesophagus and is not secreting mucus or enzymes 98.
The cardia and the rest of the stomach, on the other hand, are covered with
mucus secreting columnar epithelium. The cardia also contains mucus
producing glands. The glands are composed almost entirely of mucus
secreting cells. Few pepsinogen producing zymogenic or chief cells may be
present. Gastric or fundic glands are located in the fundus, the greater part
of the body and the proximal part of the antrum. The glands are lined with
mucus producing cells, zymogenic cells, argentaffin cells which produce
gastrin that stimulates release of gastric juice, and parietal cells. The latter
produce hydrochloric acid and intrinsic factor for the uptake of vitamin B 12.
Pyloric glands, which contain mucus producing cells and zymogenic cells,
are located in the distal part of the antrum and pyloric canal.
The mucus produced by glands and epithelial cells protects the underlying
tissue from the corrosive mixture of HCl and pepsins. The lowest pH is
measured in the distal part of the stomach, especially in the antrum, where
the mixing takes place. The main function of HCl is considered to be the
provision of a non-specific barrier against a variety of infectious organisms
since luminal pH values of 1-2 can be detected 78, 121, 163.
Emptying of the stomach into the duodenum is influenced by several facts
(Table 2.1).
Table 2.1 Factors influencing the emptying of the stomach 163.
i
Gastric motility.
ii
Volume, since the greater the volume of the gastric content the faster
the emptying.
iii
Hypertonic contents are released slower than isotonic contents.
iv
The presence of fats or fatty acids and acid in the duodenum inhibits
gastric emptying.
v
Cold liquids (4C) are emptied slower then warm liquids.
15
2.1.4 The small intestine
In humans, the small intestine is 4-5 m in length. The duodenum makes up
the first 25 cm, the jejunum refers to the next 2 m and the remainder
constitutes the ileum. There is, however, no clear boundery between the
regions. The digesta from the stomach pass into the duodenum where they
are mixed with the secretions from the liver. The duodenum contains only
one type of gland, the glands of Brunner that secrete mucus and
bicarbonate. The release of bicarbonate is increased by the introduction of
gastric juice in the duodenum. Bicarbonate is also released into the
duodenum with pancreatic juice and secreted by the epithelial cells.
Bicarbonate neutralises the digesta coming from the stomach and raises the
pH to above 5. In the jejunum, the pH gradually rises to slightly above 6, in
the mid ileum the pH becomes more neutral, pH 6.88 121.
In addition to HCO3-, pancreatic juice also contains a mixture of enzymes,
or their precursors, to hydrolyze carbohydrates, proteins and lipids. It also
contains several enzymes capable of hydrolysing minor food constituents,
e.g. nucleotides 163.
Bile is excreted into the duodenum from the liver. The major organic
constituents are bile acids, phospholipids and cholesterol which aid in lipid
digestion, and bilirubin which gives faeces their characteristic colour.
Major electrolytes in bile are K+, Na+, Cl- and HCO3-.
One of the major functions of the small intestine is the absorption of
nutrients. The small intestine is well adapted to this function. The surface
area is enhanced some 600 times by morphological structures such as the
villi and the circular Kerckring folds that are dominant in the jejunum. The
villi are finger like structures that project into the intestinal lumen and are
about 1 mm high. There are 10-40 villi per square millimeter. The luminal
surface of the villi is lined with epithelial cells, referred to as enterocytes.
The enterocytes further enlarge the available surface area since the
membrane presented to the lumen is covered with microvillous structures
approximately 1 m high and 0.1 m in diameter. The membrane is
appropriately called the brush-border membrane 163. Several
oligosaccharidases, peptidases and phosphatases are bound to the brushborder membrane, 115. These enzymes are expressed by mature
enterocytes located towards the tip of the villi 76. The main functions of
the enterocytes are digestion and the uptake of nutrients. Enterocytes are
covered by a protective mucus gel (section 2.2) that is between 5 and
400 m thick 1, 22, 134.
At the base of the villi are the crypts of Lieberkühn. These are simple
tubular glands containing mostly undifferentiated cells as well as mucus
secreting goblet cells, lysozyme and cryptdins producing Paneth cells 56
and endocrine cells. The undifferentiated cells divide and differentiate to
16
become the enterocytes lining the villus as they progress out of the crypt
towards the villous tip, as on a conveyor belt. In humans, it takes about 6
days for cells to migrate from the base of the crypt to the tip. In many other
mammals, this process takes from 1 to 3 days.
Due to uptake of nutrients and water, the volume of the digesta is gradually
reduced. Because of this reduction in volume, and fewer and slower
peristaltic contractions, the movement of the digesta is slower in the ileum
than in the upper part of the small intestine. For humans, the overall transit
time in the small intestine is 4-6 hours 188.
2.1.5 The large intestine, colon and caecum
In humans, the colon is about 1.5 m long, depending on the length of the
person. It is devided into four regions; ascending, transverse, descending
and sigmoid colon. The caecum is a blind pouch, connected to the proximal
part of the ascending colon, adjacent to the ileo-caecal valve, which
connects small and large intestine. The mucosa of the large intestine differs
from the that in the small intestine, there are no villi and the crypts of
Lieberkühn are deeper. The crypts do not contain Paneth cells, but have
more goblet cells. A few goblet cells can also be found between the
enterocytes.
The motility of the colon is quite slow. A contraction wave slowely pushes
the contents foreward at 1-2 cm.min.-1 78, 163. Because the contents do
not flow through the colon as in a prop-flow reactor and because
considerable back mixing takes place, especially in the ascending colon. A
net forward movement of about 5 cm.h-1 is often noted 163, resulting in a
transit times of up to 60 hours 188.
2.2 Mucus
Mucus is lining the epithelium of the intestine. It is an important site for
colonisation of both normal microflora and potential pathogens, and has
been used as substratum for adhesion studies in papers II-V.
2.2.1 Function
Intestinal mucus is a viscous substance produced by goblet cells and
overlying the epithelial surface. The proposed functions of mucus are many
(Table 2.2).
17
Table 2.2
i
ii
iii
iv
v
vi
vii
Some of the proposed functions of mucus 22, 60.
A barrier against chemical and proteolytic damage.
Protecting the epithelium from mechanical damage.
Lubrication, to facilitate the passage of the gut contents.
Stabilising the micro-environment at the intestinal wall.
Stabilising the association of some bacteria.
Preventing the association of others.
An energy source for the normal microflora.
2.2.2 Composition
Mucus is a complex mixture that is predominantly water (95% or more).
Large glycoproteins, mucins, are responsible for the viscous nature of
mucus and make up 0.5-5%. Mucin monomers have a Mr of approximately
250 K and consist of a protein core that makes up some 20% by weight,
and carbohydrate which constitutes some 80% by weight. Between 40-70%
of the aminoacids are threonine, serine and proline. The central part of the
protein is glycosylated and contains many tandem repeat sequences (each
repeating unit being 6 to 169 amino acids in length, dependent on the
particular mucin) 113, 126, 191. About 70% of the hydroxyl groups of
serine or threonine are in O-glycosidic linkage with N-acetylgalactosamine.
The oligosaccharide units are bound to this N-acetylgalactosamine and
radiate out like the brisles of a bottle-brush 191. The dominant sugars are
N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose and sialic
acid. The number of monosaccharides linked to GalNAc rarely exceeds 18,
and the chain may be branched. If the terminus of the chain consists of GalGlcNAc disaccharide, it carries a bloodgroup determinant 22, 21, 60, 126.
Sialic acids give mucin its negative charge, pKa=2.6 123. The highly
glycosylated part is relatively resistant to proteolysis. This part is flanked
by less glycosylated, “naked”, regions. These regions are sensitive to
proteolysis and may contain hydrophobic regions to which bacteria may
bind, by non-specific hydrophobic interactions 21, 181.
Two models exist for the polymeric structure of mucin.
i) Four mucin monomers are bound to a central ‘link’ peptide, like the sails
of a windmill, by disulphide bridges between the “naked” regions and the
‘link’ peptide 142. The size of the ‘link’ peptide has been suggested to be
70 K 142, 90 K 124 and 118 K 154, 156. The peptide makes up about
25% of the total mucin protein and 10% of the total mucin carbohydrate.
Mannose constitutes 10% of the ‘link’ peptides carbohydrate 156. The
existence of the ‘link’ peptide is very much debated, and according to some
workers 179 it does not exist.
18
ii) The other model proposes a flexible thread structure, where mucin
monomers are linked end-to-end via disulphide bridges.
Electron microscopic results, favor the latter model. They show 0.2-4 m
long threads which are neither branched nor star-shaped 155, 173.
Lipids constitute up to 40% of the dry weight of intestinal mucus 178.
About half of the lipids present in mucus are neutral lipids (free fatty acids,
cholesterol, cholesterol esters, mono, di- and triglycerides). Glycolipids
(glyceroglucolipids and glycosphingolipids, from exfoliated cells)
constitute approximately 45%. The remaining lipids (about 5%) are
phospholipids 178. The lipids in mucus affect bacterial adhesion by
modifying the hydrophobic interactions between a bacterium and its
substratum. Lipids, especially cholesterol esters, also influence the
viscosity and elasticity of mucus 127.
Free proteins make up 0.5-1% of the mucus. Part of these proteins are
perfused plasma proteins, of which albumin, IgA and IgG are the most
prevalent 45. Some proteins may originate from shedded enterocytes 35,
196. Other proteins are secreted, these include secretory IgA, lysozyme,
lactoferrin and proteinase inhibitors 21, 45, 116. Extracellular matrix
molecules, particularly fibronectin, have also been shown to be present in
mucus 179.
Salts constitute approximately 1% of mucus. Small amounts of free amino
acids, carbohydrates and nucleotides that are most likely from lysed host
cells in particular leucocytes, are also present 21, 45, 129.
2.3 The normal microflora
The microbes characteristically found at different sites of the body of
healthy subjects, are refered to as the normal microflora 188. They will
colonise specific habitats of the body surfaces and contents, and stay there
until they are displaced by organisms better adapted to occupy the habitat
66. In addition to these colonising microbes, various transient microbes
can be observed. Since they just pass through the tract, unless some factor
induces a change in the normal microflora whereby the transient microbe
can establish, their influence on the intestinal ecosystem is variable. The
normal microflora has also an important function blocking the colonisation
by potential pathogens, more about that in section 4.2.1.
2.3.1 The mouth
The mouth is one of the few places where the normal microflora is directly
involved in physiological damage and disease. Due to the different tissues
and great range in the redox-potential, a variety of habitats exists. The
19
normal microflora can adhere to many different substrata. Bacteria also
adhere to each other (coaggregation), forming a bacterial mass known as
plaque.
Some of the most common genera found in the oral cavity are:
Actinomyces, Lactobacillus, Streptococcus, Bacteroides, Prevotella,
Fusobacterium, Neisseria and Veillonella. Yeasts of the genus Candida are
also commonly found 136, 188.
2.3.2 The oesophagus
Lactobacilli and other, unidentified, Gram-positive rods and cocci have
been found to colonise the oesophagus of many animals 188. In humans,
aerobic organisms were found to be present in all cases, while anaerobic
organisms were present in 80% of the cases and yeasts in 12% 177. It has
been suggested that the organism found in the oesophagus originate from
the oropharynx and ingested food 71.
2.3.3 The stomach
In humans, the stomach normally contains only small numbers of
microorganisms. Up to 104 cfu.ml-1 gastric content has been reported 188,
and many of these are in fact transient. They originate from food and the
oropharynx 177. The microorganisms found are usually aciduric species;
lactobacilli, streptococci and Candida albicans. A high percentage of
people are colonised by Helicobacter pylori. Its natural habitat appears to
be the mucus covered non-acid-secreting epithelium of the antrum. Many
factors combine to induce H. pylori cells to change to become pathogenic
after many years of being a commensal 110. In pigs, the secreting
epithelium and especially the squamous epithelium of the pars oesophagus
are colonised by lactic acid bacteria 38, 68. Desquamation of the pars
oesophagus epithelium is supposed to inoculate the stomach contents with
lactic acid bacteria, thus ensuring their dominance 7, 68, 98, 188.
Unfortunately this theory has not been tested by surgical removal of the
pars oesophagus. It could explain why animals that do not possess a pars
oesophagus, for example humans, have no resident microflora in the
stomach 121, 177 and lower numbers of lactobacilli in their intestine
188.
2.3.4 The small intestine
In humans, the duodenum appears not to have a permanent microflora. The
microflora is affected by the pH of the stomach and duodenum, also the
20
swift flow of the digesta reduces the chance for colonisation, 103-104
cfu.ml-1 content can be observed 177, 188. The composition of the normal
microflora resembles that of the stomach 71. In the upper jejunum, up to
105 cfu.ml-1 and in the lower jejunum up to 107 cfu.ml-1 were observed by
Macy and co-workers 121. Most of these microbes were anaerobes. Due
to the slower passage of the digesta in the ileum, colonisation occurs, with
bacterial populations of 107-108 cfu.ml-1 being observed 177, 188. The
composition of the microflora in the ileum resembles that of the colon.
Major constituents are lactobacilli, enterococci, members of the
Enterobacteriaceae family, e.g. E. coli, and the obligate anaerobes,
Bacteroides, Veillonella and Clostridium 121, 188.
2.3.5 The Large intestine
Due to the slow flow rate, the colon is the main site of microbial
colonisation in humans. About 1010-1011 bacteria per gram contents are
present 163, 188. The number of bacteria increases one order of a
magnitude from proximal colon to distal colon 119. Over 40 genera and
400 species have been isolated and identified from the faeces of individuals
on several countries 166. Anaerobes out number aerobes by a factor 1000,
due to the low oxidation reduction potential. It is interesting to note that
some oxygen is available in the colon, because it diffuses from the
epithelium and is present in the lumen where as much as 1.8% O 2 has been
reported 163. The major genera found in the colon are: Bacteroides,
Bifidobacterium, Eubacterium, Ruminococcus, Peptostreptococcus,
Bacillus, Clostridium and Lactobacillus 163, 166, 188, 177. These
bacteria have a fermentative metabolism in the large intestine. Dietary
polymers, principally polysaccharides, and endogenous macromolecular
polymers, mainly mucus, that reach the colon can be hydrolysed by the
majority of the colonic bacteria and serve as an energy and carbon source.
The major metabolic end products are short chain fatty acids. These fatty
acids, especially butyric acid, are a major energy and carbon source for the
enterocytes lining the colon. They provide an estimated 10% of the daily
energy need in humans 165.
2.4 Enteric pathogens
2.4.1 General
A pathogen can be defined as a disease producing organism 147. One
should bear in mind, however, that any microorganism that can sustain
itself in an other organism (e.g. human), may occasionally cause disease
21
and this will be influenced by the health status of the host 59. In order to
be able to cause disease, a microorganism can possess so-called virulence
factors. These are special properties which distinguish potential pathogens
from harmless strains 93.
Adhesion of pathogenic E. coli has been the main subject of study for this
thesis. Its virulence factors will be discussed in more detail in the next
section (2.4.2). Adhesion of other entropathogens has also been tested, but
has usually been compared to the results obtained for E. coli strains.
2.4.2. E. coli
Most pathogens are “one-disease” organisms, however, E. coli is able to
induce a variety of diseases. Except for diarrhoea, many common diseases
are caused by E. coli 93. E. coli is the most common cause of urinary tract
infection 93 and neonatal sepsis 64. The E. coli strains that are able to
cause disease, can possess many different virulence factors. They possess
flagella (H-antigen), which make it motile, and fimbriae which allow it to
adhere to host tissues and also confer host specificity (section 5.3).
Fimbriae or fimbriae-mediated adhesion, is also thought to affect
membrane structure and integral proteins, imparing water and electrolyte
absorption and ATPase activity 19, 167, 168. Some E. coli strains can
excrete enterotoxins in the intestine. Two different kinds exist; a heat labile
toxin (LT) and a heat stable toxin (ST) 159.
Two different types of LT exist. LT-I has a high degree (75%) of amino
acid sequence identity with choleratoxin, and LT-II which so far has been
found primarily in E. coli strains isolated from animals, has not been
associated with disease. LT-I consists of five B subunits and one A subunit
which is enzymatically cleaved in A1 and A2 subunits. Unlike V. cholerae,
E. coli does not excrete LT-I. It possibly leaks out of the periplasmic space
in the presence of bile acids and trypsin or in the absence of iron. The toxin
activates adenylate cyclase which raises the cAMP level in the epithelial
cell. Raised cAMP levels cause, among other things, excretion of Na + and
K+ into the lumen. Water will follow the electrolytes due to osmotic
activity 159, 162.
ST is a family of toxins with a low molecular weight and contain 17 to 31
amino acids. They are compact and contain 3 disulphide bonds and are
therefore not easily inactivated by heating. The heat stable toxins are
subdivided into methanol soluble, ST-I (STa) and methanol insoluble, STII (STb) forms. ST-I activates guanylate cyclase which raises cGMP level.
Two ST-I subfamilies exist: ST-Ia (STp), from bovine, porcine and human
origin; and ST-Ib (STh) which is of human origin only 69. A rise in
cGMP level leads to a similar loss of electrolytes and water as with raised
22
cAMP levels. ST-II functions differently from ST-I and has so far only
been found in E. coli strains from porcine origin 159, 167.
Other virulence factors are the capsule (K-antigen), lipopolysaccharide
(endotoxin, O-antigen) and haemolysins. Capsules loosely coat the
bacterial cell and are composed mainly of carbohydrate with some
aminoacid or lipid components. Capsules protect the bacterium from the
hosts defence mechanisms, complement activation and phagocyte mediated
killing 93, 159. Almost without exception, SfaII expressing E. coli cells
possess a K1 capsule which is a sialic acid polymer 104. Sialic acids do
not trigger an antibody response because they are also present on the hosts
cells 159.
LPS makes up the outside of the Gram-negative outer membrane. It
consists of core polysaccharide, the O side chain and lipid A which is the
hydrophobic part that anchors LPS in the membrane. LPS is thought to
protect the bacterium against the hosts complement system 93, 147. It is
also responsible for many pathophysiological effects; inflammation, blood
coagulation, fibrinolysis and hypotension 147.
Two types of haemolysin can be produced by E. coli; secreted haemolysin and cell bound -haemolysin. Haemolysins form pores in the
membranes of cells, not only erythrocytes. It is thought that the haemolysin
releases iron from erythrocytes, disrupts phagocyte function and is directly
toxic to host tissues 93.
23
3 Milk
One of the characteristics of mammals is the production of milk with which
they suckle their young. Milk not only provides nutrition, but also a range
of factors that protect the newborn from many enteric pathogens. Of the
4000 or so mammalian species, the milk of only 5 or 6 species are used by
man. In addition to human milk, bovine milk is most widely used 62.
3.1 Composition and functions
Milk can easily be devided in four fractions, namely, fat, cells, casein and
whey. Milk whey has been studied in this thesis. Fat and cells can be
separated from the milk by centrifugation. The cells which include
macrophages, neutrophiles, lymphocytes 188 and cell debris will form a
pellet while the fats will collect at the top. Milk fat is concentrated in
globules with a diameter of 1-10 m. The globules are surrounded by a
membrane, rich in mucins 169, 175. These membranes will also collect in
the top layer. Casein can be separated in two ways. It can be precipitated by
lowering the pH to about 4.6 which is the isoelectric point of casein.
Alternatively, it can be coagulated by hydrolysis of -casein with proteases.
Whey, is the solution left after removal of casein 130.
Whey prepared according to the former method was used in paper IV, and
whey prepared according to the latter method was used in paper V. Minor
differences in the composition of both types of whey exist. Lactose makes
up around 70% of the dry matter in the whey from cow milk. Only 11-12%
of whey from cow milk is protein 128.
3.2 Milk whey
3.2.1 Oligosaccharides
The main sugar present in both human and cow milk is lactose. The
concentration in human milk (55-70 g.l-1) is about one and a half times
higher than in cow milk. Human milk also contains a wide range of other
oligosaccharides (3-6 g.l-1), while only traces are detected in cow milk
107.
N-acetylglucosamine (GlcNAc) containing oligosaccharides have been
shown to act as “growth factors” for esp. Bifidobacterium bifidum var.
pennsylvanicus 144. This sugar is rather rare in cow milk. This could
explain the some times observed difference in the number of bifidobacteria
in the faeces of breast fed v.s. formula fed infants. Formulas often contain
transgalactosylated
oligosaccharides.
Fermentation
of
these
24
oligosaccharides or GlcNAc in the large intestine, lowers the pH and
creates an unfavourable environment for a number of pathogens. 107,
188.
Many of the oligosaccharides naturally occurring in human milk are
thought to function as soluble receptors for pathogenic bacteria and viruses
(see chapter 6 on inhibition of adhesion).
3.2.2 Proteins
In bovine milk, whey proteins represent 20-25% of the total milk proteins.
The major whey proteins are -lactoglobulin, -lactalbumin,
immunoglobulins and blood serum albumin. Minor proteins are: lactoferrin,
lysozyme, lactoperoxidase, 2-microglobulin, free secretory component etc.
62
a) -lactoglobulin
-lactoglobulin (-lg) represents approximately 50% of the whey proteins
in cow milk. Human milk does not contain -lg. Bovine -lg originating
from the diet can be observed in human milk 90.
The monomeric molecular weight of -lg is approximately 18 000. In milk,
-lg normally exists as a dimer. It is proposed that the biological function
of -lactoglobulin (-lg) is that it binds retinol, thus protecting it against
enzymatic oxidation and facilitating its absorption. If that is in fact the
physiological function of -lg, one must question how retinol is protected
and how efficiently it is absorbed from human and rodent milk that lack
this protein 62. At present, attention is being focused on the fact that -lg
may be one of the causes of cow milk allergy 63. In paper IV and V, it is
shown that -lg also appears to have antimicrobial activity, because it
inhibits S-fimbriae mediated adhesion, see also section 6.3.6.
b) -lactalbumin
-lactalbumin (-la) is the next most prevalent whey protein and
constitutes 20% of all whey proteins in cow milk. In human milk there is
considerably more since -lactalbumin is part of the lactose synthetase
complex. It modifies the action of -galactosyltransferase so that lacose is
produced. The complex catalyses the addition of galactose to glucose to
produce lactose. -la contains a Ca2+-ion which could provide protection
against thermal denaturation 185. Although removal of the Ca2+-ion
results in denaturation of -la at approximately 50C 62, this is not likely
to be a biological function because milk will not be exposed to such
25
temperatures under natural conditions. It is more likely Ca2+ functions as a
second messenger, regulating the catalytic properties of the lactose
synthetase complex 185. -la has also been shown to be a growth
promoter for certain bifidobacteria 144.
c) Immunoglobulins
Bovine colostrum contains up to 100 g.l-1 immunoglobulins, but the
concentration decreases to less then 1 g.l -1 within a week. Human milk
contains approximately 10 g.l-1 immunoglobulins after two days, this
decreases to some 2 g.l-1 after two weeks 62. Three classes of
immunoglobulins are found in milk; IgA (the main immunoglobulin in
human milk), IgG (the main immunoglobulin in cow milk) and IgM. IgG is
monomeric, IgA dimeric and IgM pentameric. The monomers consist of
four polypeptide chains, two heavy (Mr 50-70 K) and two light (Mr 25 K),
linked by disulphide bridges. The di- and pentamers are made by linkage of
the monomers with a protein called the J-chain. IgA contains a secretory
component, which facilitates the transport of IgA from the blood to the
extracellular side 62, 184. The role of the immunoglobulins function is to
provide passive immunization for the newborn, mainly by IgA and IgG
52. An entero-mammary circulation has been described, in which
antibody-producing cells from the maternal intestine migrate to the
mammary glands. They produce secretory antibodies against enteric
pathogens common in the maternal-newborn environment and are provided
to the newborn in the milk 46. The presence of anti-idiotypic antibodies,
antibodies that “look” like the antigen, in human milk has been suggested.
This might result in even an active immunization of the newborn 83.
d) Serum albumin
Both human and bovine milk contain around 0.4 g.l -1 serum albumin.
Bovine serum albumin has a calculated molecular weight of 66 267 Da, it
has an overall ellipsoidal shape, with an axial ratio of about 1:3. In the
circulation, serum albumin transports insoluble hydrophobic molecules like
bilirubin and fatty acids, possibly in hydrophobic pockets that can open and
close 52. Cations, especially Cu2+ and Ni2+ can be bound on the surface
62.
No specific function for serum albumin in milk has been found thus far,
although its fatty acid binding ability may aid lipolysis. Serum albumin
concentrations in blood are some 10 times higher than in milk. It may leak
out of the circulation into the milk 62.
26
e) Minor whey proteins
Most of the previously mentioned minor proteins appear to have some
antimicrobial activity.
Lactoferrin is a glycoprotein with a molecular weight of about 80 000. It
can bind irreversibly two Fe3+ per molecule. This iron binding activity has
been reported to form the basis for lactoferrins antimicrobial activity 52,
62 (see section 6.2.1). It removes the iron necessary for growth of most
bacteria, with the exception of lactic acid bacteria. The highly cationic
nature of lactoferrin could be responsible for another antimicrobial effect of
lactoferrin, namely damage of the outer membrane of Gram-negative
bacteria 117, 199. Cow milk contains 0.02-0.35 mg.ml-1 lactoferrin and
this is 10 to 100 times lower than the concentration in human milk 62.
The antimicrobial activity lysozyme has been discussed in section 2.1.1 for
lysozyme in saliva.
Lactoperoxidase is a glycoprotein with a Mr of approximately 77 500. It is
a mammalian peroxidase found in saliva, tears and milk. It catalyzes the
oxidation of thiocyanate (SCN-) by hydrogen peroxide, generating OSCNand possibly also HO2SCN and H3OSCN. Lactoperoxidase makes up about
1% of whey protein. The substrates, thiocyanate and H2O2 are derived from
glucosinolates and cyanogenic glucosides, and produced by lactic acid
bacteria 70, 148. Structural damage and changes in bacterial membranes
due to exposure to OSCN- have been reported 100. However, the main
bacteriostatic effect is the contribution to blocking of the glycolysis. It is
proposed that it inhibits glucose transport, hexokinase activity and
glyceraldehyde 3-phosphate dehydrogenase activity due to oxidation of
sulfhydryl groups in metabolic enzymes. The latter enzyme appears to be
the primary target 20.
2-microglobulin (lactolin) has a Mr of 12 000 and is structurally similar to
immunoglobulins 62. It is thought to stabilize the tertiary structure of
histocompatibility antigens or is necessary for processing and intracellular
transport of the antigen 79.
Secretory component is a glycoprotein with a molecular weight of 74 000.
It not only acts as a receptor for IgA to be secreted but has also been found
to inhibit the adhesion of enterotoxigenic E. coli 74 (see section 6.2.1).
27
4 Lactobacilli
4.1 General background
The genus Lactobacillus includes regular, non-sporing and Gram-positive
non-motile rods. Since lactobacilli are unable to synthesize porphyrins,
they usually lack catalase and cytochromes, unless the porphyrins are
present in the growth medium. They are “aerotolerant” anaerobes, are
fastidious and have a fermentative metabolism, producing lactic acid as the
major metabolite during sugar fermentation. Lactobacilli are aerotolerant
and grow optimally under slightly acid conditions 6, 147.
Although their name suggests that lactobacilli are associated with milk,
they can be found in a wide range of other nutrient rich environments such
as plant matter and meat as well as being part of the normal microflora of
the mouth, intestine and vagina of mammals 6, 147. Although lactobacilli
are members of the normal microflora of the intestine (section 2.3), it
should be noted that they are not numerically dominant, and that
approximately 20-25% of humans do not have lactobacilli in their intestine
166, 188.
4.2 Proposed functions in the intestine
At the turn of the century, Metchnikoff suggested that oral ingestion of
beneficial bacteria could interfere with harmful bacteria. He regarded the
normal microflora of the large intestine as extremely harmful 188, 187.
Today it is aknowledged that the normal microflora performs an important
protective role against disease (see section 4.2.1). Lactobacilli are thought
to improve the stability of the intestinal microflora, thereby having a
beneficial effect on the health of the host 157. This use of
microorganisms, is also called probiosis. The term probiotic has been
defined as a live microbial feed supplement which beneficially affects the
host animal by improving its intestinal microbial balance 66, although it
is used today as food supplements as well. Many beneficial properties have
been proposed for probiotics and lactobacilli in particular. Some of these
are listed in Table 4.1.
In order to exert one or more of these beneficial effects, the probiotic strain
has to be present at the site where it is required. It would therefore be
desirable for the strain to colonise the host at that site. That could prove to
be difficult since in a host all possible colonisation sites could already be
occupied by members of the normal microflora or established pathogens.
The probiotic strain should have to replace a member of the normal
microflora, which is highly unlikely (see next section; 4.2.1) 164. In a
host with a disturbed microflora, this might be more feasaible. If the
28
probiotic strain can not colonise, it may have to perform while “passing
through”. In the upper gastro intestinal tract, this may work due to the
lower numbers of the normal microflora 158.
Table 4.1 Some of the proposed beneficial properties of probiotics 40, 67,
84, 137, 161.
Property
Proposed mechanism
Anti carcinogenic activity
stimulation of the immunesystem,
inactivation of mutanogens and
carcinogens, reduction of enzymes
implicated in carcinogen production.
Prevention of
deconjugation of bile acids.
hypercholesterolaemia
Improved nutritional status of vitamin production, predigestion of antifoods
nutritional factors, metabolism of lactose.
Stimulation of the immune
lactobacillal cell walls act as adjuvants.
system
Alleviation of constipation
improved intestinal motility.
Prevention of osteoporosis
Prevention of diarrhoea
Improved growth rate of farm
animals
increased bioavailability of minerals.
stabilising the normal intestinal production
of antimicrobials, blocking of adhesion
sites, inactivation of enterotoxins,
colonisation resistance (see also section
4.2.1).
Suppression of sub clinical infection by
growth depressing organism.
4.2.1. Colonisation resistance
Lactobacilli and other microorganisms may prevent diarrhoea by producing
components that are antagonistic towards pathogenic bacteria. Many
mechanisms are involved in this antagonism.
The normal microflora is considered to have a protective function against
disease. This was demonstrated by the finding that treatment of mice and
guinea pigs with antibiotics eliminated part of the normal microflora. This
loss made them extremely sensitive to infection by Salmonella enteritidis
and Vibrio cholerae (ID50, 106 bacteria for untreated animals v.s. less then
ten bacteria for antibiotic treated animals) 85. Even more convincing was
the finding that germ-free guinea pigs could be killed by as little as ten S.
enteritidis cells, while it required 109 cells to kill an animal with a normal
microflora 36.
29
The protection provided by the normal microflora is referred to by different
terms: Environmental resistance, colonisation resistance, premonition,
infection immunity, antagonism, bacterial interference, microbial
interference or competitive exclusion [187]. In this thesis, I will use the
term colonisation resistance.
In the intestine, microorganisms are competing with each other and in some
cases with the host. For the latter situation there is competition for
nutrients, not only carbon sources but also for scarce nutrients such as iron
[187]. Microorganisms are competing with each other for sites, including
sites on the mucosal surfaces [85]. Microorganisms can more successfully
compete by producing antimicrobials, either as metabolites (e.g. volatile
fatty acids, hydrogen peroxide etc.) or by the production of compounds
refered to as bacteriocins. Some strains of lactic acid bacteria have been
found to produce bacteriocins. Klaenhammer 101 defined four distinct
classes of bacteriocins from lactic acid bacteria. The typical bacteriocin,
however, is a small amphiphilic protein with antimicrobial activity that is
generally specific for closely related strains 91. This is also a potential
disadvantage as they might work against other beneficial strains instead of
against unrelated pathogens 161. Bacteriocin producing Lactobacillus
strains can be isolated from faeces. It has also been shown that E. coli cells
that produce a bacteriocin referred to as colicin, can colonise volunteers
better and longer than non-colicinogenic E. coli cells 163. It is, however,
not known whether bacteriocin production is important physiologically
[187, 188]. To answer this question, one could use a method similar to that
used by Blom and Mörtvedt 13 for the demonstration of bacteriocin
production in sausage. These workers used a bacteriocin producing strain
together with an isogenic non-bacteriocin producing, bacteriocin sensitive
strain.
In paper I it is shown that the growth inhibiting activity of acidic
metabolites from L. fermentum 104 can be enhanced by components with
an Mr over 1000. Spent culture fluid from L. fermentum 104 was
bactericidal to ampicillin resistant E. coli strain 1107. This activity could
not be inactivated by heat or protease treatment, but was found to be pH
dependent (Fig. 5 paper I). These results are typical findings for growth
inhibition by short chain fatty acids 161. Spent culture fluid fractions with
molecular weights less than 500 and 1000 were, at best, bacteriostatic (Fig.
1 and 2 paper I), however, fractions with molecular weights more than 500
or 1000 did not show bactericidal activity. These findings suggest the
presence of high a molecular weight component which is not bactericidal
by it self, but which may enhance the activity of the acidic metabolites
present in the spent culture liquid or be activated by them. This hypothesis
is strengthened by the observation that casaminoacids restore the
bactericidal activity of the <500 fraction. Enhanced bactericidal activity of
30
organic acids has been observed before. Milk and lysed horse blood were
found to greatly enhance the bactericidal activity of acetic acid against
Salmonella sp. 31.
31
5 Adhesion
5.1 General background
Adhesion to a surface is important to bacteria in most environments. It
enables them to colonise environments under conditions where they
otherwise would be washed away 201.
For example in the small intestine the flow is rather high due to secretion of
fluids into the lumen, and peristaltic movements (1-2 cm.s-1163). In
humans, transit times of 4-6 hours have been reported 188. In addition,
epithelial cells with attached bacteria are shed, and mucus is released into
the lumen continuously. Adhesion to the intestinal mucosa is regarded as a
prerequisite for colonisation of the small bowel by both pathogens and the
indigenous microflora.
5.2 Non-specific adhesion
Non-specific interactions were defined by Busscher and Weerkamp 18 as
interactions due to overall macroscopic surface properties, as charge or
surface free energy. Non-specific adhesion is the most common form of
adhesion in nature. It involves non-covalent bonds and hydrophobic
interactions.
Non-covalent bonds are:
i Electrostatic forces, which are weak in water due to its high relative
permittivity. Multivalent cations have been found to enhance adhesion
30, 17. They can form a bridge between the bacterium and the host
cell by electrostatic interaction to both.
ii Hydrogen bonds are also weakened by the presence of water.
iii Finally, van der Waals forces. This type of bonding works at distances of
over 50 nm, but has a sharp maximum at approximately 0.5 nm 184.
Due
to
plasma
membrane
phospholipids,
teichoic
acids,
lipopolysaccharides, acidic sugar residues and other factors, the net surface
charge of both bacteria and host epithelial cells is negative, thus causing a
repulsion between the cells. The so-called DLVO theory 50, 194
describes the interaction between particles of the non-covalent forces (see
Fig. 5.1). It predicts that two surfaces of similar charge may attract each
other by long-range (>50 nm) forces, created by fluctuating dipoles. At 1020 nm there exists an energy minimum. Adhesion in this so-called
secondary energy minimum is rather weak and reversible. At shorter
distances, the repulsive forces become stronger due to the overlap of the
diffuse electrical double layer (adsorbed ions on the surfaces) of both
particles. The electrostatic repulsion is strongly influenced by the ionic
32
strength of the solution (that is the ion valence and the concentration). The
higher the ionic strength, the thinner the electrical double layer and this
results in less repulsion. When the repulsive forces are overcome, the
attractive forces become stronger again and create a primary energy
minimum. The forces in this primary minimum are stronger than in the
secondary minimum and binding is irreversible.
Kinetic energy is required to overcome the electrostatic repulsion of the
diffuse electrical double layer between both minima, however, under
normal conditions, bacterial cells do not have enough kinetic energy 95.
Fimbriae (discussed in section 5.3) on the bacterial surface are much
thinner (3-10 nm) than the bacterium itself (0.5-1.5 m). They have a
smaller radius of curvature and therefore have less surface that is parallel
with the substratum. Therefore, they experience less repulsion. The
fimbriae can overcome the electrostatic repulsion and span the distance
between the primary and secondary minimum. They can bind in the
primary energy minimum, often by more specific interactions, thereby
anchoring the bacterium firmly and irreversibly to the host tissue.
Enteropathogenic E. coli appear to bind directly in the primary minimum,
possibly by inducing changes in the brush border membrane, rather than by
fimbrial attachment 53.
Figure 5.1 Attractive and repulsive forces working upon a particle
(bacterium) approaching a solid surface, according to the
DLVO theory 50, 194.
Hydrophobic interaction is mainly entropy-based. Water molecules around
a hydrophobic surface are more ordered then around a hydrophilic one as
they can not make many hydrogen bonds at a hydrophobic surface.
33
Interactions between two hydrophobic surfaces in an aqueous solution,
therefore liberates the ordered water molecules originally surrounding the
surfaces. This increases the entropy and reduces the free energy of the
system 28.
Craven and co-workers 43 found that non-specific binding, especially
hydrophobic interaction, plays an important role in in vitro binding of
Salmonella sp. to chicken mucus and enterocytes. Adhesion of
Streptococcus sobrinus and Streptococcus sanguis to saliva coated
hydroxyapatite has also been found to be non-specific 133. Hydrophobic
interaction was found to play an important role in the adhesion of marine
yeasts irrespective of the hydrophobicity of the substratum 193. Adhesion
of E. coli HB101 to HeLa cells was also found to be dependent on surface
hydrophobicity of the bacteria 41, as does the adhesion of Yersinia
enterocolitica to brush border membranes 150. Breines and Burnham 16
reported that they could reduce non-specific adhesion to uro-epithelial
cells, by treating the cells with 0.4 M HCl. In fact, this treatment would
hydrolyze mucus normally covering the cells and thereby reduce specific
adhesion to the mucus rather than reducing non-specific adhesion as
proposed. In table 4 (paper III) it can be seen that many of the tested strains
bind as well to BSA as to porcine ileal mucus. The observed adhesion to
BSA is regarded as non-specific adhesion. Infact, BSA is generally used as
blocking agent in binding studies, e.g. 15, 75, 109. In this thesis, the term
non-specific adhesion is used to refer to adhesion which is not a specific
receptor-adhesin interaction, as described below. Although non-specific, in
the in vivo situation, non-specific adhesion may play a more important role
than generally believed.
5.3 Specific adhesion
After the bacterium has bound rather loosely to the host cell surface by
non-specific interactions, specific interactions may develop 9. Specific
adhesion typically involves a lock-and-key bond between complementary
receptor and adhesin molecules. An adhesin is a structure on the surface of
a microorganism, and a receptor is a complementary structure on the
surface of a host cell 9.
The adhesin molecule has to be presented in such a way that it can easily
interact with the receptor and not be affected by interfering molecular
structures and negatively charged molecules present on the bacterial
surface 96. Many members of the Enterobacteriaceae family express
fimbriae. Fimbriae had been observed by several workers, but some
thought them to be artifacts. Houwink and van Iterson 87 were the first to
suggest that they might be involved in the adhesive ability of certain
34
strains. Duguid and co-workers then showed that these structures were
involved in haemagglutination and adhesion, and suggested their biological
function to be the binding to cells and solid particles in the intestine 54.
Fimbriae were shown to be fibre like structures (hence their name) made of
polymers of polypeptide units, arranged around a central canal. Each
bacterial cell possess several hundreds of fimbriae, which are spread
peritrichously over the cell and are anchored to the outer membrane. They
are approximately 0.3 to 2.0 m long and 3 to 10 nm thick 55, 93. The
adhesive molecule can be situated on the tip of the fimbria, as with P
fimbriae and CFA/I 17, 49, 96, both on the tip and laterally, as with type
1 fimbriae 102, or laterally as with K88 and K99 fimbriae 49. The
receptors proposed for the fimbrial adhesins that were used in this thesis are
presented in table 5.1.
Except for K. oxytoca type 3 fimbriae, all adhesins bind to carbohydrate
moieties. Carbohydrate-binding proteins usually contain a hydrophobic
cleft with aromatic and charged residues projecting into it. The former
interact with the carbohydrate backbone and the latter with the
hydroxylgroups of the carbohydrate 106. This has been found to be true
for fimbriae too. Many fimbriae are hydrophobic 49 or possess
hydrophobic pockets, like type 1 fimbriae 93, 102 and P fimbriae 93. In
addition, P fimbriae make five hydrogen bonds with a polar ridge of
Gal(1-4)Gal 106. So, the same non-covalent bonds which play such an
important role in non-specific binding, are also responsible for the
interactions in specific binding although at a much shorter range and
therefore requiring the right stereochemical conformation to guarantee
optimized fit 138. Even though the binding of each fimbria is rather weak,
a bacterium possesses several hundreds of fimbriae which together attach
the bacterium firmly to the substratum.
Binding of enteropathogens to carbohydrate moieties is not surprising.
Epithelial cells lining the intestinal mucosa have a glycocalyx on the
surface. This glycocalyx provides a surface rich in glycoproteins and
glycolipids. Glycolipids and especially glycoproteins present in mucus
and/or on epithelial cell membranes, are considered to be receptors for
pathogens. For many types of fimbriae, several glycoprotein receptors have
been isolated. It is not unlikely that one adhesin has many different
receptors, however, it could also reflect differences in affinity by different
fimbrial serotypes, differences in glycoprotein from porcine and murine
origin or from erythrocytes 145, and possible proteolytic degradation
prior to isolation 49, 96.
35
Table 5.1 Fimbrial adhesins discussed in this thesis and their proposed
receptors.
Bacterium/Fimbrium
Receptora
Escherichia coli K88
-D-Gal, fucose58, GalNac, GlcNac3,
Gal(1-3)Gal 197, Galactosylceramide15
E. coli K99
GalNac(1-4)Gal(1-4)GlcCer, NeuGc(23)Gal(1-4)Glc(1-1)Cer140, 180, 189
E. coli F41
GalNac, GlcNac114
E. coli 987P
Fucose, Glucose, Galactose, Mannose,
corresponding amino sugars and N-acetylated
derivatives 48
E. coli, Salmonella
Mannosides
typhimurium Type I
E. coli CFA
GalNac(1-4)Gal(1-4)GlcCer, Gm2
ganglioside, sialoglycoprotein 145
E. coli Sfa
NeuAc(2-3)Gal(1-3)GalNAc103, 141
Yersinia enterocolitica
Mucin carbohydrate moiety125, extracellular
Yad A
matrix proteins
S. typhimurium
Gal(1-3)GalNAc73
Salmonella enteritidis
Fibronectin 37, plasminogen 176
thin aggregative fimbriae
Klebsiella oxytoca
non-carbohydrate segments of type V
type 3 fimbriae
collagen32
a
Gal = Galactose; GalNAc = N-acetyl galactosamine; Glc = Glucose;
NeuGc = N-glycolyl neuraminic acid; NeuAc = N-acetyl neuraminic acid;
Cer = Ceramide; GlcNAc = N-acetyl glucosamine
Recognition of different receptors by different adhesins allows bacteria to
bind to specific tissues, e.g. urinary tract epithelium, different epithelial
cells in the gastrointestinal tract 8, dental surfaces, other bacteria
(coaggregation) etc. It also allows host specificity, e.g. E. coli CFA/I and
CFA/II infections are limited to humans, E. coli K88 fimbriated cells infect
pigs, E. coli K99 infections are limited to calves, lambs and piglets 49.
The K99 fimbriae are less host specific.
5.4 Adhesion assays
In vitro adhesion assays are used to investigate the adhesion of bacteria to
various substrata. Haemagglutination was the first assay used to study the
adhesive properties of fimbriated bacteria 54, 55. Subsequently, tissue
36
pieces 97, tissue culture cells 47, epithelial cells 43, 198, brush border
membranes 172, immobilised mucus 35, 108, 120 and immobilised
ileostomy glycoproteins paper III, IV and V have been used as substrata
for adhesion. In principle, the substrata and bacteria are incubated together
to allow binding to occur. Unattached cells are removed by washings,
centrifugation or buoyancy 75. The number of bound bacteria can be
determined by microscopy 47, colony forming units 183, radiometry
108, spectrophotometry 120, ELISA 160 and other enzyme linked
assays 75.
In this thesis, the Caco-2 cell line, immobilised porcine ileal mucus and
immobilised ileostomy glycoproteins were used as substrata for adhesion.
Human ileostomy glycoproteins that were a generous gift from Dr. J.G.H.
Ruseler-van Embden (Erasmus University, Rotterdam, The Netherlands),
were used as a model for human intestinal mucus. Adhesion of bacteria was
determined radiometrically 35, 108. In short, mucus or ileostomy
glycoproteins were passively immobilised by overnight incubation at 4C
to polystyrene microtitrewells. Before use, wells were washed to remove
excess mucus that was not immobilised. The Caco-2 cell line was grown
under standard conditions and used after two weeks, when the cells were
well differentiated. Caco-2 cells differentiate in vitro and express several
properties that are characteristic for mature enterocytes. In order to study
components that inhibit adhesion, the substrata were pre-incubated with test
solutions, namely Lactobacillus spent culture fluid, bovine colostrum whey
or their fractions. As controls the substrates were pre-incubated with
uninoculated medium or buffer phosphate buffered saline (PBS), HEPES
buffered Hanks salt solution (HH-buffer). After 1 hour at 37C the wells
were washed and a suspension of radiolabelled bacteria was added. After
incubation at 37C for 1 hour, the substrates were washed to remove
unbound bacteria. Bound bacteria were released and lysed by incubation at
60C with SDS and NaOH. Activity was determined by liquid scintillation.
The extend of inhibition of adhesion was determined by comparison of
radioactivity in wells treated with test solution as compared to the
radioactivity in wells treated with buffer or uninoculated medium as the
control.
37
6 Inhibition of adhesion
6.1 General background
As stated in the previous chapter, adhesion can play an important role in
colonisation of the small intestine by microbes. Adhesion of a pathogen to
host tissue is one of the first events in the development of many infectious
diseases and is considered a prerequisite for infection 9, 59, 93, 152.
Consequently blocking the initial attachment could effectively interfere
with bacterial infection at an early stage 59. Adhesion could be inhibited
by either affecting the adhesin on the bacterial cell or the receptor on the
substrate.
Phagocytes also carry receptors on their surface which allow them to
recognize and bind to a variety of bacteria. Alternatively, bacteria can be
opsonised with complement components and immunoglobulins, which
subsequently bind to phagocyte receptors 153. After this binding, the
bacteria are readilly phagocytosed and killed as part of the host defence
mechanism 147, 89. It could be undesirable to interfere with this
adhesion, except in the case of HIV 135. HIV binds to the CD4 receptor
of T4 lymphocytes where after it infects the cell.
6.2 Affecting the adhesin.
6.2.1 Blocking the adhesin.
The adhesion to the substratum can be influenced by the presence of
soluble receptor analogues. These analogues will bind to the adhesin and
block its binding site, making it impossible to bind to the real receptor on
the substrata. The adhesive capacity of the bacteria will thereby be
impaired. Intestinal mucus 65, 120, 156, Tamm-Horsfall protein
(uromucoid or urinary slime) 93 and urinary oligosaccharides 99 are
proposed to prevent in situ binding by functioning as natural receptor
analogues. They block the binding site of the adhesin and potential
pathogens would then be unable to bind to the underlying epithelium.
Carbohydrate components that function as receptor analogues have also
been tested in vivo and it has been proposed that this would reduce the
concentration of K88 fimbriated E. coli 186. In this study, even though
the E. coli K88 numbers decreased, the concentration of total E. coli was
also lower in the test group, while the ratio of K88 fimbriated E. coli to
total E. coli was unchanged. This suggests that a mechanism other than
blocking of the adhesin by the carbohydrate receptor analogue may have
been involved.
38
Cranberry and blueberry juice have been found to inhibit the adhesion of
type 1 and P fimbriated E. coli to eukaryotic cells 200. Using an in vitro
adhesion assay it was concluded that the active components were
condensed tannins. The components bind to the bacterial surface, possibly
to the adhesin itself 139. Clinical studies indicated that cranberry juice
significantly reduced the incidence of urinary tract infections 5. It remains
to be determined if the condensed tannins that were found to be the active
component in the in vitro assay, are responsible for the in vivo effect.
Sanchez and co-workers 160 found that a range of glycoproteins were
able to inhibit adhesion of F17 fimbriated E. coli to bovine mucus and
brush-border membranes. The glycoconjugates were proposed to work as
receptor analogues, however, no proof was given that the glycoconjugates
did not affect the immobilised mucus or brush-borders. Although the
bacteria were preincubated with the glycoconjugates, the mixture was
incubated with the immobilised mucus or brush-borders in the presence of
the glycoconjugate.
Milk has been found to contain substances that inhibit the adhesion of
pathogens. These include immunologic components, glycoproteins and
oligosaccharides. In one study 42 it was shown that oligosaccharides that
inhibited adhesion of a urinary tract pathogenic E. coli were also detected
in urine from both mother and the infant. Similar oligosaccharides were
also present in the breast milk.
As immunoglobulins provided in the milk are usually directed against a
pathogen or its components, immunoglobulins affecting the adhesion of
pathogens, will usually affect the adhesin. Fimbriae are strongly
immunogenic antigens. Vaccination of dams with K88 antigen induced the
presence of K88 specific IgG in the colostrum. The colostrum inhibited
markedly the adhesion of K88 mediated adhesion to porcine intestinal
tissue, however, levels fell progressively in the milk at 2 and 7 days postpartum. High levels of K88 specific IgG were also found in the serum,
suggesting that vaccination with purified adhesin may be an option for
inhibiting pathogen adhesion 97. Vaccination of pregnant sows with
purified 987P fimbriae, gave high protection to piglets upon challenge with
enterotoxigenic E. coli expressing homologous 987P fimbriae. In the
control groups, 88.6% of the piglets developed diarrhoea v.s. 18% in the
vaccinated group 143. A long term disadvantage of vaccination may be
the selection for new fimbrial types 195. Cravioto and co-workers 44
reported that secretory IgA from human milk bound to an enteropathogenic
E. coli adherence factor, thereby inhibiting its adhesion in a tissue culture
cells. Free secretory component (see section 3.2.2e), from human milk, has
been found to inhibit haemaglutination of CFA/I fimbriated E. coli 74. It
was suggested that the oligosaccharide residues of free secretory
39
component may inhibit E. coli adhesion. Some of the saccharides found on
free secretory component are galactose, mannose, fucose, glucosamine and
sialic acid 149. There are reports that these components can function as
receptors for many fimbrial adhesins (see Tab. 5.1).
Numerous non-immunological factors have been found to block adhesion
of enteropathogens. In many cases the activity has glycoproteins and
oligosaccharides, which may act as soluble receptor analogues. S-fimbriae
mediated adhesion was found to be inhibited by mucins in human milk
169, 170, 171. It should be noted that these mucins differ considerably
from intestinal mucins 154. Unidentified glycoproteins in human milk
have been proposed to work as soluble receptor analogues against E. coli
4, 86, V. cholerae 86 and Y. enterocolitica 92. Human milk contains a
large quantity and a great variety of free oligosaccharides (see section
3.2.1). Many of these oligosaccharides have been identified as receptors for
pathogenic microorganisms 44, 107.
Lactoferrin has antimicrobial activity due to its iron sequestering activity,
and inhibits the invasion of HeLa cells by E. coli expressing the Yersinia
pseudotuberculosis invasin gene. Although lactoferrin was found to bind to
the tissue culture cells, it was concluded that the binding to E. coli was
responsible for the activity by disrupting the outer-membrane 116, 117,
199. It has also been suggested that oligosaccharide residues of lactoferrin
were responsible for blocking of E. coli CFA/I binding 74 and type 1
mediated adhesion 190.
Finally, a non-specific blocking of adhesion has been reported using bovine
casein 133. It was proposed that the casein coated the S. sobrinus and S.
sanguis cells, thereby changing the surface properties of the bacteria and
inhibiting the adhesive capacity.
6.2.2 Reducing adhesin expression.
A more direct way of influencing the adhesin, is reducing its expression.
This has been shown to be possible for both Gram-positive and Gramnegative bacteria by using sub-lethal concentrations of antibiotics.
Although antibiotics can affect slime production by bacteria and induce
changes in the bacterial peptidoglycan structure, it is suggested that only
the latter changes would affect the adhesive capacities 24. Changes in the
peptidoglycan could induce changes in surface hydrophobicity and charge
118, 182.
Exposure to sublethal concentrations of antibiotics can also influence
fimbriae. Electron microscopy has been used to show that cells were
partially or totally devoid of fimbriae after exposure to antibiotics 51.
Subsequently, Breines and Burnham 16 showed presence of fimbriae, on
40
bacteria exposed to antibiotics, with the help of monoclonal antibodies
against fimbriae. They argued that this inconsistency can be explained by
the presence of fimbrial stumps, or by the formation of fragile fimbriae
which can have broken off during preparation for electron microscopy. On
the other hand, Eisenstein and co-workers 57 showed that significantly
longer fimbriae are present on the bacteria, suggesting that the fimbriae are
not particularly fragile. These workers suggested that fimbriae may be
produced that lack an adhesin.
Conway and Adams 39 showed that the food color erythrosine was able
to inhibit adhesion of Lactobacillus sp. They concluded that erythrosine
binds to the bacterial cell surface and alters bacterial metabolism, thereby
preventing production of bacterial adhesin.
6.3 Affecting the receptor
While affecting pathogen adhesion by affecting the receptor seems
desirable, one must also consider that in so doing, one may interfere with
another function of the receptor molecule since it is likely to be involved in
biological processes, which may be disturbed.
6.3.1 No receptor.
Specific adhesion, mediated by adhesins, requires the presence of a
receptor. Presence or absence of a receptor may determine susceptibility or
resistance, respectively, to a particular pathogen. As stated above, E. coli
K88 infections are limited to pigs, however, not all pigs are susceptible to
colonisation by E. coli K88.
Two phenotypes of porcine enterocyte brush-borders have been identified,
namely brush-borders that carry a receptor for K88 fimbriae and those that
do not. The presence of the receptor was shown to be the dominant trait
while absence of the K88 receptor was a recessive trait. At least 4 porcine
phenotypes have been demonstrated since they bind K88ab and/or K88ac
and/or K88ad fimbriae 12, 172.
6.3.2 Modifying the receptor.
Adhesion can be inhibited by modifying the receptor in such a way that it
no longer can specifically interact with the adhesin. Trypsin treatment of
receptor containing intestinal material inhibited the interaction between the
material and the K88 adhesin 131. This suggests that proteolytic activity
may have a protective function against microbial colonisation of the small
intestine 27. Proteolytic treatment with bromelain, has been shown to
reduce the receptor activity for K88 fimbriae in vivo 27. It could therefore
41
function as a prophylactic treatment against E. coli K88 induced diarrhoea
132. Protease activity from Saccharomyces boulardii has also been
suggested to remove or reduce brush border glycoproteins involved in
adhesion of pathogens ot the mucosa, analogous to the inhibition of
Clostridium difficile toxin A binding 146.
Displacement of receptors has also been observed 133. Bovine casein
derivatives displaced human serum albumin from saliva coated
hydroxyapatite, thereby interfering with the adhesion of S. sobrinus and S.
sanguis. The displacement can be explained in terms of the Vroman effect,
which describes the displacement of adsorbed proteins by more surface
active ones.
6.3.3 Specific blocking of the receptor.
A receptor can be blocked specifically by addition of free adhesin. The
adhesin will bind to the receptor, blocking it from any interaction with
adhesin carrying pathogens. Outer membranes have been shown to be
competitive inhibitors of E. coli adhesion to epithelial cells 174.
Lipoteichoic and teichoic acid have been found to inhibit adhesion of
Staphylococcus aureus to epithelial cells by binding to the staphylococcal
receptor site 2. Lectins with specificity for different carbohydrates have
been found to inhibit the adhesion of E. coli to brush-border membranes.
Interestingly, the inhibition was independent of the carbohydrate specificity
and solely depended upon the capacity of the lectin to bind to the brushborder 94. One could postulate that the lectin may not bind to the
receptor, but inhibits the adhesion by steric hindrance.
6.3.4 Colonisation resistance. Non-specific blocking of the receptor.
The mechanisms which together account for the phenomenon of
colonisation resistance (as described in 4.2.1) will make it difficult for a
new microorganism to establish. Interfering with the adhesion of the new
coming microorganism can be done most effective by blocking its potential
receptors, either specifically or non-specifically, i.e. steric hindrance. This
concept has been extensively investigated for lactobacilli. It has been
shown that lactobacilli and some other organisms [88] inhibit the adhesion
of pathogens by blocking their receptors.
Reid and co-workers investigated the effect of the normal microflora and
lactobacilli on the adhesion of uropathogens to uroepithelial cells. They
found that pre-incubation of uroepithelial cells with Lactobacillus sp. or a
diphtheroid organism isolated from the normal microflora were able to
inhibit the adhesion of Gram-negative uropathogens to uroepithelial cells in
suspension 25. They also achieved complete or partial inhibition of
42
adhesion of Gram-negative uropathogens using bacterial cell wall
fragments isolated from a Lactobacillus strain, although whole viable cells
were more effective than cell wall fragments. It was concluded that
lipoteichoic acid was responsible for the adhesion of Lactobacillus cells to
uroepithelial cells, but that steric hindrance was the major mechanism of
adhesion inhibition 26. In a later study, however, it was shown that steric
hindrance is not likely to be the sole mechanism of action. Of equally
adherent Lactobacillus strains, the larger strain, which should cover a larger
surface area of the host cell, did not cause the largest inhibition of
adhesion. Adhesiveness appears to be a major determinant in adhesion
inhibition 151.
Heat killed Lactobacillus acidophilus has been found to inhibit the
adhesion of enteropathogens to tissue cultures. Fourniat and co-workers
61 found that the spent culture liquid, pH adjusted to 5, was necessary for
the adhesion inhibitory effect. The liquid itself had no effect on the
adhesion of enteropathogenic E. coli. Since lysed lactobacilli were not able
to inhibit adhesion, it was concluded that the inhibition was not a specific
competition for a common receptor but rather steric hindrance of binding
sites for the E. coli. Servin and co-workers 10, 11, 29, 33, 34 found that
heat killed and live L. acidophilus cells and bifidobacteria were able to
inhibit the adhesion and invasion of enteropathogenic E. coli, S.
typhimurium, Yersinia pseudotuberculosis and Listeria monocytogenes to
tissue culture cells. They too concluded that blocking of the receptors by
steric hindrance is the most likely mechanism. However, all experiments
were done in the presence of culture liquid and no mention is made as to
whether the pH was adjusted. It is also not clear what control was used in
the experiments since it could have been treatment with fresh broth, buffer
or may not have been treated. Activity was found to be dose-dependent,
however dilution of the bacterial suspension with fresh broth is likely to
change the pH of the suspension. It has been shown that pH is a very
important factor in adhesion to tissue culture cells 77, 112.
6.3.5 Lactobacillus cell wall fragments. Non-specific blocking of the
receptor.
As mentioned above, whole lactobacilli, live and heat-killed are able to
block adhesion of enteropathogens to tissue culture cells. Cell wall
fragments have also been found to inhibit enteropathogen adhesion 26.
Blomberg and co-workers 14 found a component in Lactobacillus spent
culture liquid that inhibited the adhesion of K88 expressing E. coli cells to
porcine ileal mucus. It was concluded to be a heat-stable protein with a
molecular weight of more than 250K. The mechanism of action was
proposed to be blocking of adhesion by steric hindrance through
43
interactions with mucus components. The phenomenon was studied in more
detail as described in papers II and III.
Lactobacilli were grown in a semi-defined medium 105, with a high
acetate content, namely 15 g.l-1 sodium acetate. The activity of the spent
culture liquid was tested in an in vitro adhesion assay as described in
section 5.4. It appeared that acetate was essential for the presence of the
component in the spent culture liquid. Acetate also caused a rapid decline
in the number of viable cells in the culture. The K88 adhesion inhibitory
component could also be isolated first at late log-phase, no stationary-phase
was observed (Fig. 1 paper II). It can therefore be speculated that the active
component originates from dead Lactobacillus cells. It is apparent that
acetate which is added to Lactobacillus selective media to inhibit growth of
other microbes also creates conditions which have negative effects on
lactobacilli. The activity was found in fractions corresponding to a size of
approximately 1700K when dialyzed and concentrated spent culture liquid
from L. fermentum 104r was fractionated by gel filtration. When the
concentrate was treated with pronase prior to fractionation, the size was
reduced to approximately 1100K. This indicates that protein could be
associated with the component but appears not to be necessary for activity.
After pronase treatment, only small amounts of protein were found to be
present in the fractions, but considerable amounts of carbohydrate.
Previously the activity had been found to be heat stable (15 min. 80C)
14. In fact, the activity was found to be extremely heat stable with no loss
of activity after 20 min. at 121C. The fractions were found to contain
glucose, N-acetylglucosamine and galactose, carbohydrates typically found
in Lactobacillus cell wall 111. Treatment with lysozyme and glucose
oxidase, but not endo--galactosidase, removed the activity. These findings
suggest that the active component is of carbohydrate nature, and very likely
to be a cell wall component. Because cell wall fragments from
homogenized cells were not active and because the activity could not be
removed from the spent culture liquid by centrifugation, 27000 x g, 1.5h
14, it was concluded that the active component was soluble in nature. Of
the cell wall components, it appeared not to be teichoic acid since the
activity was found to be acid stable. Treatment with 0.05 M HCl at 60C
for 20 min. hydrolyses teichoic acids, the adhesion inhibiting activity was
however not affected by this treatment 138. The adhesion of E. coli K88
to immobilised mucus pretreated with Lactobacillus spent culture liquid
was found to be 9.23  2.81 (n = 3) as compared to the medium treated
control. Spent culture liquid treated with HCl reduced the adhesion to a
similar level 5.97  1.85 (n = 3).
The precise mode of action still remains to be explained. Using
ellipsometry measurements, it was not possible to detect any change in
44
thickness of the immobilised mucus film upon treatment with spent culture
liquid. Further more this treatment did not produce any change in
hydrophobicity of the immobilised mucus film. When material in the spent
culture liquid was radiolabelled, a small part of the activity was found to
bind to the immobilised mucus. Preliminary results from Scatchard plot
analysis, using radiolabelled spent culture liquid, suggest binding to a
single receptor. Spent culture liquid appeared to inhibit the adhesion to
certain ileal mucus fractions considerably more than to other fractions (Fig.
5 paper II). A preliminary conclusion is that the active component binds to
certain mucus components and blocks the adhesion by steric hindrance.
This hypothesis is further supported by the finding that spent culture liquid
from L. fermentum 104r was able to inhibit adhesion mediated by all K88
fimbrial serotypes and mediated by CFA/II and SfaII fimbriae, the latter
two being human pathogens (paper III). The adhesion of the target strains,
CFA/I, CFA/II, CFA/IV and SfaII fimbriae, was inhibited by spent culture
fluid from strain HBL8 more than by spent culture fluids from the other
tested Lactobacillus strains . Spent culture liquids from all tested
Lactobacillus strains were able to inhibit SfaII-fimbriae-mediated adhesion.
(paper III).
What argues against the steric hindrance theory is the finding that adhesion
to mucus is also inhibited when mucus is treated with spent culture liquid
as a suspension prior to immobilization. When mucus was first
immobilised and then treated with, 5x concentrated, spent culture liquid, as
in all previous cases, adhesion of E. coli K88 was 20.57%  2.76 (n = 3).
When mucus was first incubated with, 5x concentrated, spent culture
liquid, as a suspension, and subsequently immobilised, adhesion of E. coli
K88 was found to be 24.68%  2.47 (n = 3). Consequently steric hindrance
can only occur if the active component and E. coli bind to the same
molecule but to different epitopes. Blocking of the K88 receptors during
immobilization is not likely, since this was performed at 4C and no
significant activity could be observed at 0C (paper II).
It was also observed that many other lactobacilli of enteric origin were able
to inhibit K88ac adhesion. Strains from porcine origin performed best, but
even strains from human origin were able to block this adhesion. Host
specificity appears not to be of major importance for this property. Strains
from non-enteric origin were not able to cause any inhibiton of adhesion of
E. coli K88ac (paper III). For in vivo application of this substance, in situ
production of the substance would be preferable. This is most likely to
occur for a strain originating from the same host since it is more likely to
colonise and therefore grow in situ. Unfortunately, in vivo production of the
inhibitory component may not occur since, after growth in porcine ileal
mucus no inhibitory activity could be observed. This could be linked with
the fact that high concentrations of acetate are necessary.
45
Another possibility for in vivo use is the direct oral administration of the
substance. This should preferably be administered prophylactically, since
already bound E. coli are not released. If used when the enteropathogen is
already present, it may limit the proliferation of the pathogen. In an attempt
to mimic the intestinal situation, porcine ileal mucus was immobilised on
glass beads in a column. After washing, the column was filled with spent
culture liquid and incubated at 37C for 1,5 h. Fractions were collected and
tested for K88 adhesion inhibiting activity (Figure 6.1), the inhibitory
activity was found to be significantly reduced. The fractions were
concentrated and applied to a Sepharose CL 4B column. The carbohydrate
pattern was found to be slightly changed (Figure 6.2). These findings
indicate that the mucus is affecting the activity present in spent culture
liquid. As mentioned previously, mucus contains lysozyme. The lysozyme
may have reduced the activity. Furthermore, the active component may
have bound to the immobilised mucus. Consequently, it may be concluded
that large amounts of active component are probably necessary for in vivo
activity in order to saturate the mucus and/or compensate for loss of
activity due to lysozyme activity. Administration of the substance has the
advantage that the best producing strain can be used, whereas if the live
microbe is used one would need a strain which can colonise the host.
140
Adhesion (%)
120
100
80
60
40
20
0
spent
culture
liquid
2
4
Fraction (1ml)
6
Figure 6.1 Adhesion of Escherichia coli 1107 K88ac to mucus treated with
Lactobacillus fermentum 104r spent culture liquid fractions
from a column with immobilised mucus.
46
110
100
Carbohydrate ( g/ml)
90
80
70
60
50
40
30
20
10
0
-10
0
10 20 30 40 50 60 70 80 90
Fraction (2ml)
Figure 6.2 Carbohydrate profile of Lactobacillus fermentum 104r spent
culture liquid () and L. fermentum 104r spent culture liquid
from a column with immobilised mucus (). Fractionation was
performed using Sepharose CL 4b.
6.3.6 -lactoglobulin. Non-specific blocking of the receptor.
As mentioned above, many milk components have been found to inhibit
adhesion of a variety of enteropathogens, in most cases by blocking the
adhesin of the bacterium. In this section the effect of milk components, in
particular -lactoglobulin, on the host receptor will be discussed.
Giampaglia and Silva 72 showed that pretreatment of HeLa cells with
human colostrum inhibited the adhesion to these cells of all tested E. coli
strains, by 37 to 90%, depending on the strain tested. Simultaneous
incubation of E. coli and HeLa cells in the presence of human colostrum or
milk, was found to be more effective, with the adhesion of E. coli inhibited
by up to 97%. Oligosaccharides and sIgA were suggested to be the active
components, albeit no attempt was made to identify them or the mechanism
of inhibition.
As mentioned in section 3.2.2a, the major function of -lactoglobulin (-lg)
is proposed to be the binding of retinol. In paper IV and V it is shown that
-lg also appears to have an antimicrobial activity.
47
Bovine colostrum was defatted by centrifugation and acidified to
precipitate caseins. The pH of the resultant whey was re-adjusted to neutral,
and the whey was fractionated by ultra filtration into fractions with
different molecular weights: <100K, <30K and <10K. The fractions were
tested for their ability to inhibit the adhesion of SfaII-fimbriated E. coli to
human ileostomy glycoproteins. SfaII mediated adhesion was found to be
inhibited by colostrum whey, its <100K and <30K fractions, but not by the
<10K fraction. Adhesion was reduced to approximately 25% of the buffer
treated control.
After purification, using anion exchange chromatography, an 18K protein
was found, with an isoelectric point of approximately 5.75. The amino acid
sequence of the NH2 terminus, was found to be identical to -lg. In paper
V, -lg was purified from milk whey by gel filtration.
The activity was found to be very heat stable with no loss of activity after
40 min. at 120C. It was suggested that disulphide bridges may stabilise
parts of the protein. It was hypothesized that these were important for the
observed adhesion inhibitory activity. Boiling of the protein in the presence
of a reducing agent, -mercaptoethanol, abolished the activity. Thus
supporting the hypothesis. Limited digestion of -lg by CNBr at
methionine and cysteine residues, also abolished activity, further
supporting the hypothesis that the areas around the cysteine residues are
important for the activity of -lg.
Pre-treatment of E. coli SfaII did not inhibit adhesion, however, inhibition
was noted after pre-treatment of immobilised ileostomy glycoproteins. -lg
appears not to recognize N-acetyl-neuraminyl-2,3-lactose (NANA), the
receptor of SfaII-fimbriae. Pre-incubation of -lg with NANA did not
affect the inhibitory activity. -lg, however, does bind to the immobilised
ileostomy glycoproteins, most likely by using multiple binding sites. This
was concluded from a Scatchard plot (Fig. 2, paper V) and from binding
studies to immobilised ileostomy glycoproteins fractions (Fig. 3, paper V).
Commercially available -lg was found to also inhibit SfaII mediated
adhesion, in a concentration dependent manner (Fig. 1 paper V). The
optimal concentration was found to be 10-50 g.ml-1. Similar concentration
dependent effects can be observed for antibody-antigen reactions 23,
suggesting a fixed ratio between -lg and its target. Three out of four
groups of ileostomy glycoprotein fractions that bound -lg also bound E.
coli SfaII. Consequently, it was hypothesised that -lg recognizes a
different epitope on the same molecule as E. coli SfaII, thus blocking the
SfaII receptor by steric hindrance. General steric hindrance is unlikely
since of all tested strains, only the adhesion of E. coli expressing SfaI or
SfaII and S. enteritidis and S. typhimurium was inhibited, with the adhesion
of the latter two being inhibited to a very limited extend. The hypothesis of
48
two epitopes on one molecule is strengthened by the observation that
ileostomy glycoproteins do not inhibit the adhesion of E. coli SfaII to
enterocytes when suspended with -lg (see below).
Considering the fact that -lg is present in cow milk and not in human
milk, it is surprising to find that the adhesion of the above mentioned
human pathogens was inhibited, while the adhesion of the two tested
bovine enteropathogens was not affected. Recently, 3-hydroxyphthalic
anhydride modified -lg has been found to block the CD4 cell receptor for
HIV 135, which is not a bovine pathogen.
As previously mentioned, mucus is believed to block bacterial adhesins
65, 122, 150, 156. Mucins have been found to inhibit adhesion of SfaII
fimbriae to buccal cells 169, 170, 171. It can therefore be hypothesised
that by reducing the binding between mucus and the fimbrial adhesin, SfaII
fimbriated E. coli can penetrate the mucus gel and bind to the underlying
enterocytes. This would thus promote infection. To test this hypothesis, E.
coli SfaII was allowed to bind to Caco-2 cells in the presence of HH-buffer,
ileostomy glycoproteins (≡mucus) or the combination of ileostomy
glycoproteins and -lg. Adhesion of E. coli in the presence of ileostomy
glycoproteins reduced the adhesion to 56.14%  15.44 (n=7) as compared
to the control experiment in the presence of HH-buffer. In the presence of
both ileostomy glycoproteins and -lg, the adhesion was not significantly
altered; 94.21  14.47 (n=7), compared to the control. These results
confirm that mucus does block binding to the underlying enterocytes. They
also suggest that the blocking function of intestinal mucus may be impaired
by -lg. On the other hand, E. coli SfaII may not be able to make an initial
binding to the mucus in the presence of -lg and thus be unable to colonise
and penetrate the mucus layer to subsequently cause infection.
The in vivo significance of -lg as an adhesion inhibitor remains, however,
to be assessed. -lg might be used prophylactically, or therapeutically in
combination with antibiotics to avoid further translocation.The latter would
be important if risk for antibiotic resistance exists. At present, -lg is
considered to be one of the causes of cow milk allergy 63. Also, if not
only the intestine but also the oral cavity is the site of infection 80, it
should be tested whether -lg even inhibits the adhesion to buccal cells, in
order to be effective.
49
7 Conclusions
The thesis focuses on substances that inhibit the initial binding of
pathogenic E. coli to intestinal mucus in vitro. Since adhesion is considered
to be a prerequisite for pathogenesis 9, 59, 93, 152, interfering with the
adhesion of a pathogen may prevent the establishment of disease in an
early stage.
Many substances have been reported to inhibit the adhesion of pathogens in
vitro. Most substances which are described to have an adhesion inhibitory
effect against enteropathogens, work as receptor analogues and block the
bacterial adhesin (section 6.2.1). The two substances described in this
thesis, appear to block the receptor(s) sites of the intestinal mucus. It was
proposed that the substances block the receptor(s) for the pathogen by
steric hindrance. Identification of the receptors for the adhesion inhibitory
substances would provide further information on the mechanism involved.
It was concluded that spent culture liquid from L. fermentum 104r contains
polysaccharides with an estimated Mr of 1700 K that mediate the adhesion
inhibitory activity. The polysaccharides are likely to be soluble cell wall
fragments, since the activity is affected by lysozyme and they contain
monosaccharides typically found in cell walls (paper II). The adhesion
inhibitory activity was mainly directed against the different K88 fimbrial
serotypes and SfaII fimbriae. Other Lactobacillus strains of intestinal origin
were also found to produce similar activity (paper III).
L. fermentum 104r was also found to produce a high molecular weight
substance that potentiates the bactericidal activity of the organic acids
produced by its metabolism. The in vivo significance of this effect remains
to be investigated with unanswered questions as to whether the substance is
produced in vivo and whether the in vivo concentration of organic acids are
sufficiently high to exert any growth inhibiting effect (paper I).
The substance mediating the adhesion inhibitory activity in bovine
colostrum, was identified as -lactoglobulin (-lg), a major cow milk whey
protein (paper IV). -lg was found to bind to several intestinal mucus
proteins, and to inhibit the adhesion of SfaII and, to a lesser extent, SfaI
fimbriae (paper V).
The two adhesion inhibitory substances described in this thesis, have not
been tested in vivo. Preliminary data indicate potential problems with in
vivo use. The Lactobacillus cell wall fragments are sensitive to lysozyme
(paper II). Lysozyme present in the intestine might inactivate the fragments
before they reach the site where they should exert their activity; the ileum.
Chemical modification that would protect against hydrolysis by lysozyme,
may solve this problem. -lg may be used prophylactically. -lg is,
50
however, considered to be a cause of allergy to cow milk 63, and may
therefore not be desirable to be used prophylactically.
A fundamental question to be answered is if reduced adhesion to mucus is
beneficial for the host. The ability of bacteria to bind to mucus constituents
might be advantageous for the host as it prolongs the time of mucus
penetration and may prevent pathogens from binding to the underlying
epithelium. For -lg it has been shown (see section 6.3.6) that reduced
binding to intestinal mucus may actually enhance the binding abilities of
SfaII fimbriae expressing E. coli to the underlying epithelial cells. On the
other hand, the binding may also be advantageous for the bacterium since it
facilitates colonisation of the mucus layer. In order for such colonisation to
take place, the bacterium must either multiply at a rate exceeding the rate at
which mucus is sloughed into the lumen of the intestine or actively
penetrate the mucus 65, 122, 150. The balance between these two effects
may be different for different bacterial strains.
In summary, it can be concluded that L. fermentum 104r spent culture
liquid contains substances that potentiate the bactericidal activity of organic
acids and contains polysaccharides that inhibit K88-fimbriae-mediated
adhesion. Furthermore, it was also demonstrated that -lg present in bovine
colostrum and milk, inhibits SfaII-fimbriae-mediated adhesion.
51
8 Acknowledgements
I am very grateful to my supervisor Patricia ‘Trish’ Conway, who replied
positive when I was looking for a place to do the practical part of my
undergraduate studies and invited me to continue as ‘doktorand’. I also
admire her indestructible optimism.
Also my sincere gratitude to Seppo Salminen for making my move to
Finland possible, being co-supervisor during the last two years and for
help with practical things like baby clothes and a pram.
Thanks also to Lennart Adler and Staffan Kjelleberg who were always
willing to help with lots of things during my Ph.D. studies.
Thanks to all present and previous members of ‘mag-tarm gruppen’ () for
the pleasant atmosphere to work in and all the small talk: Allan, AnnCathrin, Anna, Annika, Camilla, Christer, David, Elisabeth, Lars, Maurilia,
Paul, Ruth, and especially to Agneta; for your help with lots of things even
when I was in Finland, and Lena and Anders; for all the valuable things
you have told and learned me concerning intestinal microbiology, when I
was still a novice.
Thanks also to the members of ‘bacterial virulence’; Elise, Jarmo, Maria,
Pia, Reija, Saija and Yasmin and of course Miikki for the discussions and
your good sense of humor. And to the people at Viable Ltd., in particular
Ari; for the non-sense discussions we’ve had, and Elina; for introducing me
into the mysterious world of cell culture and for being a pleasant colleague
and friend.
I also wish to thank all the people who helped me with the practical and
bureaucratic things involved in moving abroad; Anita, Maureen, Lise and
Inga in Göteborg and Kaija in Åbo/Turku. But most of all for their good
spirit, which positively affects the respective departments.
52
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