Download The ecological digestive system and its colonisation

Rev. sci. tech. Off.
int. Epiz.,
1989, 8 (2), 259-273.
The ecological digestive system
and its colonisation
D.C. SAVAGE *
Summary: The gastro-intestinal tract of animals of most species is colonised
by an indigenous microflora consisting of micro-organisms able to grow
anaerobically. Most of the organisms are aero-intolerant anaerobic bacteria.
These organisms colonise the tract through successions in infants, culminating
in climax microbial communities in adults. These communities colonise habitats
which, depending upon the animal species, may be located in the lumen, on
the epithelial surface or deep in the crypts of Lieberkühn in any part of the
tract. A variety of factors regulate which species can form communities in
habitats in animals of a given species. Some of these factors derive from the
animal host, its diet and environment; some derive from the various microbial
species interacting with each other. One important factor is the capacity of micro­
organisms to colonise epithelial surfaces. We are studying certain properties
of micro-organisms able to colonise gastric or intestinal epithelial surfaces in
laboratory mice and rats. The properties under study probably evolved in the
organisms to enable them to adapt to and colonise their surface habitats. Our
approach involves using molecular and genetic technology to identify specific
macromolecules that are important in allowing the organisms to remain in and
fill a niche (function) on the epithelial surfaces.
K E Y W O R D S : Gastro-intestinal tract - Indigenous microflora - Intestinal
epithelium - N o r m a l microflora - R o d e n t s .
INTRODUCTION
The ecological digestive system can be defined with reference to four elements.
These include (a) the animal host and its environment; (b) the gastro-intestinal tract
and its associated organs; (c) the host's ingesta, digesta and faeces; and (d) the
indigenous gastro-intestinal microbiota. These elements interact in complex ways,
with each able to influence the others. In this review, I focus upon the fourth element,
the microbiota, placing emphasis upon mechanisms by which indigenous micro­
organisms colonise habitats in the gastro-intestinal tract. I have focused in my own
research on the mechanisms by which micro-organisms colonise the tracts of mammals
with simple stomachs, principally laboratory rodents. The mechanisms discussed in
detail are therefore those by which micro-organisms colonise the tracts of rodents.
Where applicable, however, reference is made to mammals of other taxonomic groups,
including humans, swine and ruminants.
* University of Tennessee, D e p a r t m e n t of Microbiology, M 4 0 9 Walters Life Sciences Building,
Knoxville, Tennessee 37996-0845, U S A .
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THE INDIGENOUS GASTRO-INTESTINAL MICROBIOTA
Micro-organisms
The indigenous gastro-intestinal microbiota consists of bacteria of many taxonomic
groups as well as eukaryotic micro-organisms of a few groups (35). All of the
organisms involved can derive energy for growth in atmospheres lacking oxygen. Most
are sensitive to oxygen and are killed when exposed to it; these organisms can be
cultured in vitro only when media are poised at low oxidation-reduction potentials
and incubated in atmospheres free of oxygen. The populations of anaerobic bacteria
can be enormous, on the order of 10 cells per gram of content in some areas of
the gastro-intestinal tract. The micro-organisms colonise the tracts of newborn animals
in distinguishable patterns called successions (35).
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Successions
Successional patterns have been studied in some detail only for laboratory rodents
(11, 35, 42), chickens (35) and mammals of a few other species including humans
(28). Each succession in mammals has proved to be similar in overall pattern.
Moreover, in broad terms, the microbial genera involved are similar, particularly those
of the prokaryotic micro-organisms. Nevertheless, the succession of each animal
species is distinctive in many specific elements. Therefore, the gastro-intestinal
microflora of a mammal colonises the alimentary tract in a succession characteristic
of the animal species.
Climax biota
Succession of the microbiota ends when a climax biota has formed (35). The climax
biota in adults changes little during the lives of healthy animals maintained under
stable environmental and nutritional conditions (35, 46). As with their successions,
adult biotas are similar in general characteristics from one animal species to the next,
and microbial genera at climax are much the same for mammals of most, if not all,
species studied. Again, however, the biota of each species is distinctive in many specific
details. The climax biota of an adult animal of a given taxonomic group is therefore
as unique as its succession.
Climax communities, habitats and niches
Climax microbiotas of various animal species differ from one another in terms
of some microbial genera and many species present, and also in terms of where the
organisms form communities in the tract (35, 42). Indigenous micro-organisms
organise during succession into climax communities occupying habitats that are
essentially geographical areas in the tract. Some of these areas are of microscopic
dimension (35, 36). Microbial habitats may be located in any region of the tract, i.e.,
in the foregut (stomach), midgut (small intestine) or hindgut (caecum, large intestine).
Depending upon the animal species, habitats can exist in the lumen, on the epithelial
surface of the mucosa and even deep in the crypts of Lieberkühn in the mucosa (36).
Therefore, the microbiota of an animal of a given taxonomic group is distinctive not
only in regard to the microbial genera and species present, but also in the way those
micro-organisms are organised in communities in various regions of the tract.
The microbiota varies in detail from one animal species to the next because
ecological factors influencing the biota also vary among species (35, 36). Such factors
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regulate which microbial genera and species can form climax communities during
succession, where those communities locate, and how the micro-organisms function,
i.e., fill a niche, in the tract (36). The mechanisms by which indigenous micro­
organisms respond to ecological forces and colonise and fill a niche in a habitat are
my major concern in research and in this review.
REGULATORY FACTORS
IN T H E ECOLOGICAL DIGESTIVE SYSTEM
Allogenic and autogenic factors
Allogenic and autogenic factors regulate which microbial genera and species form
climax communities, where those communities locate and how the micro-organisms
function in them in a gastro-intestinal ecosystem (42, 45). Allogenic factors are
influences on the microbiota of the host and the host's ingesta and environment.
Autogenic factors are influences of indigenous microbial cells on each other and on
other micro-organisms. These influences interact in complex ways in the processes
regulating the biota, and the processes may differ from one habitat to another (12,
35, 38, 41).
Many allogenic and autogenic factors influence a microbiota during its succession
and climax phases. Influences exerted during succession progressively change in type
or intensity as the microbiota develops, the host matures and its diet and environment
alter (28, 35, 42, 45). These changing ecological conditions dictate the successional
pattern. As that pattern nears climax, the influences stabilise and remain generally
constant in type and intensity during the life of a healthy adult animal. This is the
case even when the adult's diet or environment changes to some extent (28, 35, 42, 45).
Consequences of disruption in the regulatory processes
Allogenic and autogenic regulatory influences can change radically when the host
is stressed in certain ways (37); starving or exposed to other forms of acute malnutrition
(37, 56); is given certain drugs, particularly antibacterial compounds (14, 37, 45);
or is affected by certain diseases, especially those that modify immunological responses
(14, 45). As a consequence, the regulatory processes in each habitat can change. The
biota may then change in regard to which genera and species are present, in where
the micro-organisms can be found and in how they function genetically and
biochemically in the tract (35, 37, 40).
Such changes can have severe consequences for the host. For example, intestinal
diseases caused by micro-organisms may develop. In adult animals whose regulatory
processes function normally, micro-organisms which cause some, if not most, intestinal
diseases are unable to colonise the gastro-intestinal tract or can colonise it only to
small population levels, often with little impact on the host's health (14, 45). Pathogens
are prevented from establishing in a habitat by the normal processes regulating the
indigenous biota (19, 45). This phenomenon has been named "colonisation resistance"
(14).
Colonisation resistance can diminish in force or be lost in its entirety when
influences regulating the biota are disrupted. As a result, microbial species with
appropriate capacities can establish in the tract and cause damage leading to disease,
including pseudomembranous colitis (32, 54) and salmonellosis (45).
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Pseudomembranous colitis in a human may develop after certain antibacterial drugs
have been used in treating that individual for other infectious conditions (32).
Salmonellosis may develop in an individual after stress of certain types, such as that
resulting from strenuous travel, or when the individual is starved or otherwise seriously
malnourished (45). As noted above, antibacterial drugs, stress of certain forms and
acute malnutrition can disrupt the processes regulating the indigenous microbiota.
An animal host may also experience nutritional problems when its microbiota is
disrupted (44). Under such circumstances, ruminant species suffer the most severe
consequences (15, 17). As is now well known, adult ruminants are dependent for their
nutrition upon their ruminai microbiota. In addition, nonruminant mammals of many
species also derive at least part of their nutrition from their microbiota (44). Animals
with caeca are also known to depend to varying degrees upon the caecal microbiota
for their carbon and energy requirements (44). Indeed, even humans depend to some
extent for nutrients upon their indigenous microbiota (44). Thus, when the microbiota
is disrupted, all mammals undoubtedly experience some nutritional deficiency.
Mammals with disrupted microbiotas may experience physiological problems other
than those involving resistance to disease or resulting from nutritional deficiencies
(44). It is well known that the microbiota influence many physiological properties
of the host, including absorptive processes in the intestines (61), metabolism of
cholesterol and other steroids (31), and oxidative energy metabolism (61). When the
microbiota or its functioning is altered, these physiological properties of the animal
may change as well (37). As in cases of malnutrition and loss of colonisation resistance,
the host may experience adverse consequences when physiological properties change
(37, 44). Therefore, forces that disrupt the microbiota can have many nutritional,
immunological and physiological consequences that may adversely affect the health
and welfare of the host animal.
Even though a disrupted biota can have adverse consequences for the host, little
as yet is known in detail about the mechanisms mediating the autogenic and allogenic
factors in the processes that stabilise and regulate the biota. These mechanisms are
extremely complicated and, as noted, the factors are highly interactive in the regulatory
processes. It follows that the processes are exceedingly complex in function and
mechanistic detail. Some of the regulatory factors have global functions, and exert
a major influence on the biota in habitats throughout the tract. By contrast, other
factors have restricted and focal functions, with major effects only in certain habitats
(35, 42, 45). Factors of both types may interact in a regulatory process which is unique
for a given habitat. As a consequence, the process regulating the biota in any given
habitat, even that of a microhabitat, can be understood in mechanistic detail only
by an exhaustive study. For animals of most species, even the specific habitats and
niches of micro-organisms in the indigenous biota have yet to be defined (42, 44).
Therefore, little progress has been made in understanding the process which regulates
the biota in any habitat. Some progress has been made, however, in studies of
individual allogenic and autogenic factors involved in the regulatory processes of
certain animal species.
Probably because their impact on micro-organisms in a given area of the tract
is major and therefore obvious, some global functions have been studied in detail
in recent years. Allogenic factors such as pH in the stomach and intestines (30, 35,
42), bile steroids in the small and large intestines (35) and the host's ingesta (food
and drugs) (14, 35, 42) have been extensively examined. Likewise, autogenic factors
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such as short-chain volatile fatty acids in the caecum (35, 54) and the capacity of
micro-organisms to associate with epithelial surfaces (12, 35, 36) have also received
much attention. In spite of these efforts, however, little is known as yet about how
these and other factors interact in a process regulating the biota in a given habitat.
One recent study, that of Freter and his colleagues, stands as a monument to the
difficulty of gaining information on the function of as few as two autogenic factors
in just one region of the tract of an animal of one species (12, 13). In a complex
analysis involving the use of germ-free animals, fermenters, extensive data-processing
and model simulation, Freter et al. demonstrated that two major factors regulate
certain bacterial components of the caecal biota in laboratory mice. These factors
are the capacity of the bacteria to compete in the extant environment for certain
saccharides as carbon and energy sources (59) and to associate with the mucosal
epithelium (12). Both of these factors may have global effects in the gastro-intestinal
ecosystem, but their function differs in detail according to the habitat under study.
This concept will be amplified as we now look in greater detail at just one of the
factors analysed by Freter et al. for its function in the regulatory process in the mouse
caecum, that is, the capacity of micro-organisms to associate with epithelial surfaces.
EPITHELIAL ASSOCIATION
AS A N AUTOGENIC REGULATORY FACTOR
Epithelial association in mammals
Micro-organisms of many major taxonomic classes have been found on gastro­
intestinal epithelial surfaces in mammals of numerous species. In some cases, the
organisms form thick layers on an epithelial surface. Such layers have been found
on stratified squamous epithelia and on secreting columnar epithelia. In the case of
squamous epithelia, microbial cells may adhere to the squames or to keratinocytes.
Organisms adherent to such cells form a base to which other microbial cells aggregate,
forming a layer. In the case of columnar epithelium, the microbial layers can be found
in mucous gels overlying epithelial surfaces (36, 38, 43). In such layers, the microbial
cells may adhere to receptors in the membranes of the epithelial cells (36, 38, 41,
46) or to large molecules in the mucous gel (36, 38, 46). As will be seen, however,
micro-organisms may also colonise mucous gels without adhering to any structures.
Whatever the mechanisms mediating the capacity of micro-organisms to associate
with epithelial surfaces, that capacity is a major factor influencing succession, climax
community structure and location, and microbial function in the gastro-intestinal
ecosystem (35, 42).
Gastro-intestinal epithelial surfaces and communities
With the possible exception of some surfaces lined with stratified squamous
epithelium, gastro-intestinal surfaces are dynamic three-dimensional "compartments"
(36). These compartments are composed of mucinous glycoproteins, flowing on a
glycocalyx composed of glycoproteins and glycolipids overlying the epithelial cell
membrane which is convoluted into microvilli. The membranes themselves and the
glycocalyx are presumably in a dynamic fluid state with continuous movement and
transposition of their macromolecular constituents. The compartments are made
dynamic as well by the secretive and absorptive functions of the epithelial cells and
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movement engendered by epithelial replacement, mucosal motility and peristalsis.
Micro-organisms able to associate with gastro-intestinal epithelia have evolved
mechanisms for colonising these dynamic and complex compartments.
Most of the direct evidence that indigenous micro-organisms have evolved to
colonise epithelial surface compartments has been derived from study of communities
occupying such compartments in laboratory rats and mice (36, 42). Five communites
can be distinguished in the tracts of these animals, as follows:
a) Gram-positive bacteria, principally Lactobacillus species, which colonise the
keratinising stratified squamous epithelium of the forestomach;
b) yeasts of the genus Candida which colonise the secreting columnar epithelium
of the glandular stomach;
c) filamentous bacteria colonising the epithelium of the distal two-thirds of the
small intestine;
d) facultative and strictly anaerobic bacteria colonising the epithelium of the
caecum and colon;
e) helical-shaped bacteria and spirochaetes colonising the crypts of Lieberkühn
in the caecum and colon (8, 9, 29, 36).
Each of these communities is unique in regard to the microbial species that compose
it, in the habitat it occupies and in the regulatory process that influences it. Each
will be discussed in detail with reference to the mechanisms by which the micro­
organisms associate with the epithelium in their habitat and also to some autogenic
and allogenic forces that have an impact on the organisms in their epithelial habitats.
The gastric Lactobacillus community
Cells of Gram-positive bacteria, principally certain strains of Lactobacillus species,
can be found on the squamous gastric epithelium of the newborn rodent as early as
one to two days after birth (11, 42, 48). One or two days later, layers of the bacteria
are well-formed on the epithelium; after another week, the layers are thick and
prominent. The layers persist as the animal grows (42). Most cells present in the
community adhere to each other rather than to the epithelial surface. Nevertheless,
those nearest the epithelium adhere not only to other lactobacillus cells but also to
the keratinocytes (36, 39, 46). These bacterial cells serve as hold-fasts anchoring the
community to the epithelium.
In general, only certain strains of lactobacilli, some but not all of which were
originally derived from laboratory rodents, can adhere to and colonise the gastric
epithelium (22, 57, 58). Therefore, at least some mechanisms mediating the capacity
of the bacteria to colonise the surface have evolved specifically to respond to ecological
conditions prevailing in that habitat. The capacity of the bacteria to adhere to the
keratinocytes is undoubtedly mediated by such specific mechanisms (36, 41, 46). We
have focused in our research on the molecular mechanisms by which the bacteria
adhere to the keratinised squames (27).
The mechanisms by which Gram-positive bacterial pathogens of certain species
adhere to epithelial surfaces have been studied extensively. From findings generated
in such efforts, Beachey and his colleagues proposed a molecular model for the
mechanism by which such pathogens adhered to surfaces (6). The model involved
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proteins, teichoic acids and lipoteichoic acids. These macromolecules were postulated
to interact to form aggregates of complex structure that anchored the bacterial cell
by its cytoplasmic membrane to the membrane of the animal cell (6). The "anchors"
in the membranes of both the prokaryotic and eukaryotic cells were said to be the
hydrophobic lipid ends of lipoteichoic acids intercollating into the hydrophobic lipid
bilayer of the membranes. The hydrophilic moieties of the lipoteichoic anchor
molecules were said to be bridged to each other by proteins and teichoic acids free
of their lipid moieties (6). We have used this model as a working hypothesis in studying
the mechanism by which the lactobacilli adhere to the keratinocytes.
In testing this hypothesis, we have analysed numerous strains of lactobacilli for
the capacity to produce lipoteichoic acids while growing in culture media (49). Some
of the strains were derived from rodents, and some were isolated from other animals.
A few of the rodent strains, and all strains from other animals, were unable to colonise
the gastric epithelial surface (22, 49). Many, but not all, of the strains assayed were
found to produce lipoteichoic acids under the growth conditions used. The capacity
of the strains to produce the acids could not be related, however, to a capacity to
colonise the epithelial surface. Although the molecules were detected in extracts of
cells of some of the strains able to colonise the surface, they were not detected in
a few such strains. We have therefore reached a tentative conclusion that lipoteichoic
acids may be unessential for the adherence of lactobacilli to gastric keratinocytes.
We are now focusing on study of surface proteins of the bacteria.
Specific strains of Gram-negative bacterial pathogens usually induce intestinal
disease only in animals of certain species (18). The organisms express this host
specificity, at least in part, because they have on their surfaces protein adhesives
(lectins) by which they bind to certain oligosaccharide receptors in the membranes
of intestinal epithelial cells (18). As noted above, lactobacillus strains also bind only
to the gastro-intestinal surfaces in animals of certain species (41). Host specificity
may be mediated in part by proteins able to bind only to specific receptor
macromolecules in the epithelial cells.
Proteins and polysaccharides, as well as lipoteichoic acids, can be extracted with
aqueous buffers from the surface of lactobacilli isolated from gastro-intestinal sources
(49). As assessed by Polyacrylamide gel electrophoresis, numerous proteins are present
in such extracts from lactobacillus cells able to adhere to and colonise the gastric
surface in mice (unpublished findings). We are using genetic and biochemical methods
to determine which of the proteins are the specific adhesives.
Using bacterial transformation methods, we developed from a strain unable to
colonise the gastric surface, some lactobacillus strains with the capacity to do so.
Genomic DNA was extracted from a strain able to colonise the surface and
transformed into a strain lacking that capacity. Germ-free mice were then associated
with the strain treated with the DNA. The associated animals served to select for
strains able to colonise the gastric surface (27). Our aim in developing the genetic
transformants was to focus on traits they had gained from the DNA-donor parental
strain.
Six isolates from the mice were characterised for metabolic patterns, plasmid
content, antibiotic resistance patterns and reactions with antibodies specific for
antigens on the surface of the parental strains (27). Each of them retained one or
more of the markers for antibiotic resistance and the metabolic pattern of the parental
strain unable to colonise the epithelium (DNA-recipient). They had all gained from
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the DNA-donor parent the capacity to colonise the gastric surface as well as reactivity
with the antibodies specific for antigens of that parental strain (27). We have also
recently compared the cellular hydrophobicity (60) of the transformants and the
parental strains. The transformant cells reacted in the assays at a level comparable
to that of the DNA-donor. The cell surfaces of both parent and transformants are
substantially more hydrophobic than those of the DNA-recipient (unpublished
findings). We are now focusing on proteins with hydrophobic domains isolated from
the surface of transformant cells and testing whether the proteins derived from genes
from the DNA-donor. Any such proteins will be studied as potential adhesives.
The gastric yeast community
As noted, the yeast Candida pintolopesii colonises mucous gel on the surface of
the secreting epithelium of the murine stomach (42). It also can adhere in vitro to
keratinocytes and colonise the keratinising gastric epithelium in experimental animals,
but only when the community of lactic acid bacteria normally resident on that surface
is missing (34). Moreover, when the lactobacillus community is experimentally
reestablished in animals colonised by the yeast, the eukaryotic cells can be found only
on the secreting surface. The bacteria may be able to displace the yeast from the surface
because they multiply at a faster rate than the eukaryote. However, the lactobacilli
may also be able to compete more aggressively than the yeast for adhesive receptors
(34, 36). This hypothesis remains untested pending results of our work on the
lactobacillus adhesives and epithelial receptors.
Even though C. pintolopesii can colonise the keratinising epithelium when the
lactobacilli are not present, the reverse is not true; the lactobacilli do not colonise
the secreting epithelium when the yeast community is missing (35, and unpublished
findings). The lactobacilli can grow in a culture medium adjusted to pH 4.5, but not
in medium at an acidity less than pH 4.5. By contrast, the yeast can multiply
anaerobically in a medium adjusted to pH 3.5 and aerobically at pH 2.5 (2, 16). The
contents of the lumen of the non-secreting side of the rodent stomach are acidic at
about pH 4.5, while those of the secreting side are at about pH 2.5 (21). The hydrogen
ion present in the lumen on the secreting side of the stomach may thus inhibit the
bacteria from colonising the epithelium in the area. However, ecological factors other
than acidity may also contribute to preventing the lactobacilli from establishing on
the secreting surface. More research is needed on this subject.
The secreting epithelial cells of the stomach are known to be protected from acid
in the lumen by the mucous gel that coats them. This protection is conferred by
gradients of hydrogen ion, serving as pH gradients, in the mucous gel (1, 3, 33, 50).
Therefore, the pH can be low on the luminal side of the gel but as high as pH 7.0
on the surface of the epithelial membranes. Although C. pintolopesii can grow in
culture media adjusted to pH 2.5, it multiplies at optimum rates and reaches highest
growth yields when incubated either in air or in an anaerobic atmosphere at pH 7.0
(2). Its capacity to grow most rapidly at the neutral pH suggests that it is well-adapted
for growth in its habitat, where it intimately associates with the epithelial membranes
(42).
C. pintolopesii has the capacity to grow aerobically and anaerobically (2). This
capacity also indicates that the organism is uniquely adapted to colonise its natural
habitat. Yeast cells in the murine stomach do not take up oxygen at a detectable rate
(2). Therefore, while occupying the habitat, the organism may gain its energy for
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growth primarily from its anaerobic metabolism. Its capacity to respire aerobically
cannot, however, be entirely ruled out as a source of energy in the habitat.
Bacteria of certain genera that colonise intestinal mucous gel while intimately
associating with epithelial membranes are known to multiply most efficiently in
atmospheres containing from 2 to 4% oxygen (8, 29). Such findings suggest that some
oxygen passes from the blood in underlying capillaries through the epithelial monolayer
into the mucous gel on epithelial membranes in the intestines. If the same phenomenon
takes place in the secreting stomach, then, even though we were unable to detect oxygen
uptake in C. pintolopesii cells on the epithelium, the cells may still be generating some
energy through aerobic respiratory processes. Because oxygen would be present at
only low partial pressures on the epithelium, the yeast could be respiring aerobically
at low levels which are difficult to detect by conventional methods. This hypothesis
requires further testing.
The intestinal filament community
In spite of concerted efforts by ourselves and some other investigators, the
filamentous prokaryote(s) that colonises the mucous gel overlying the epithelium in
the murine small intestine has never been grown in culture media. For this reason,
the organism has been studied only by microscopic techniques (4, 20, 42). As a
consequence, except for bowel motility and epithelial turnover, little is known about
the ecological forces which affect it in its natural habitat.
In colonising the epithelial habitat, the organism is subjected to peristalsis, villous
motility and the normal mechanisms by which the epithelium is renewed ("turns
over"). Peristalsis forces the digesta in the lumen to flow at rapid rates towards the
distal end of the intestine (35, 36). Villous motility slowly moves the mucous gel
towards the distal end as well (35, 36). During normal turnover, the epithelial cells
move along the villus from the crypts of Lieberkühn to extrusion zones at the villous
tip where they are extruded into the intestinal lumen (36).
The organisms have evolved mechanisms to overcome these ecological influences.
A specialised hold-fast cell enables the organisms to adhere to the membranes of the
epithelial cells (5, 10, 51). The hold-fast cell is located at one end of the filament
and binds by one end to the epithelial membrane forming a depression, a "socket",
in the cell (51). The membrane of the epithelial cell surrounding the socket remains
intact, but undergoes an ultrastructural change, as does the underlying cytoplasm,
which can be interpreted as a sol-to-gel transformation (51). As a consequence, the
bacterial filament tightly binds to the epithelial membrane. Indeed, the filaments are
bound so firmly that they will break off without dislodging from the attachment site
in mucosae prepared for microscopy (4). This binding is undoubtedly a powerful
mechanism by which the bacterium overcomes the influence of peristalsis and villous
motility and stabilises its community on the intestinal surface.
Because the filaments are firmly bound to epithelial cells, they transit the villi
and are extruded into the lumen along with those cells during epithelial turnover (5,
10). To be able to remain in the habitat, therefore, the organism appears to us to
have evolved reproductive mechanisms which overcome the influence of epithelial
turnover (5, 10, 40). Two possible mechanisms have been identified through
microscopic study. The first involves two bodies that develop inside each segment
(cell?) of a filament (5, 10, 40). These bodies resemble the hold-fast cell in structure,
and may be released upon spontaneous rupture of the "mother cell" inside which
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they have developed. They may be motile and attracted by Chemotaxis to the
epithelium where they attach to the epithelial cell membrane. Adequate testing of
this hypothesis must await successful culture of the filaments in artificial media.
The second mechanism by which the organism reproduces on the epithelium may
involve segments along the length of the filament which convert to hold-fast bodies
(4). The modified segments may be able to break apart from each other, freeing a
segment of the filament with a new hold-fast cell on one end. Such a segment may
be able to bind to the membrane of an epithelial cell before it is swept away by the
flow of mucous gel. This hypothesis also cannot be adequately tested until the
organism is cultured in vitro.
The intestinal anaerobe community/the intestinal helical community
As described earlier, a community consisting of strains of species of anaerobic
and facultatively anaerobic bacteria of various genera colonises the mucous gel
overlying the caecal and proximal colonic epithelium in mice and rats (42, 55). These
organisms form a dense population in the gel. Underlying that population, nearest
to the epithelial cell membranes and deep in the crypts of Lieberkühn, a community
of unclassified helical-shaped bacteria and spirochaetes also colonises the mucous
gel (47). Most, if not all, of the organisms in these communities share a distinctive
characteristic: they are motile (53). Indeed, motility is so common a trait among
members of the communities that it is undoubtedly required for colonisation. It may
be necessary for bacteria to be able to enter and move around in the mucous gel to
compete for limited nutrient resources in the dense population of mixed genera and
species (40, 53). We have been testing this hypothesis using a strain of strictly anaerobic
bacterium isolated from epithelial mucus from the caecum of a mouse (52).
Using enrichment techniques, we cultured a number of isolates of motile, strictly
anaerobic bacteria from epithelial mucus scraped from a mouse caecum. The most
motile of the isolates was characterised, found not to conform to any known
taxonomic grouping, and named Roseburia cecicola (52). R. cecicola accumulates
in epithelial mucous gel in the caecum in experimental animals (23). It is an oxygenintolerant, anaerobic bacterium and is immobilised and killed by even brief exposure
to air (52). It is highly motile and requires that motility to colonise its natural habitat
in experimental mice, but only when micro-organisms of other indigenous species,
some of which can colonise the caecal mucous gel, are also present (53). These findings
support the hypothesis that motility is essential for anaerobes in the mucous gel to
compete with other micro-organisms. It is not clear, however, whether the organisms
are competing for nutrients. That issue is under investigation.
The anaerobe's motility is mediated by a fascicle of 25 to 30 flagella located on
one pole and subpolarly on one side of the cell. We have initiated a study to determine
how the flagella function in the caecal mucous gel. In preparation, we have cloned
and sequenced the gene encoding R. cecicola's flagellin, the protein from which its
flagella are constructed (24, 26). The DNA sequence was studied by computer analysis
(DNASTAR) and the amino acid sequence of the protein was inferred. Additional
analysis with the FASTP programme (courtesy of Dr T. Joys, Department of
Microbiology, Texas Tech University Health Sciences Center, Lubbock, Texas)
revealed regions in the DNA sequence which are homologous with sequences in the
constant region of the flagellin genes of bacteria of both Gram-negative and Grampositive genera. These findings confirm that the gene cloned was indeed that of the
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roseburial flagellin gene. We are now making site-specific mutations in the cloned
gene. Such altered sequences will be used in deriving R. cecicola strains with flagella
and flagellar fascicles altered in structure in specific ways. These strains will be used
in experiments on how the altered flagella or fascicle function as the anaerobe colonises
caecal mucous gel in vitro and in vivo (7).
While cloning the flagellin gene of R. cecicola, we discovered that the organism's
genomic DNA rapidly degrades when intact or lysed cells are exposed to air (25).
Therefore, all DNA to be used for molecular manipulation and recombinant DNA
techniques must be extracted from the lysates of the cells of the anaerobe in
atmospheres free of oxygen (25). Linear bacteriophage (lambda) DNA added to such
lysates degrades along with the roseburial DNA when the preparations are exposed
to air. By contrast, covalently closed circular plasmid DNA added to comparable
lysates exposed to air is only nicked (25). The DNA's are degraded (if linear) or nicked
(if plasmid) even in roseburial lysates treated with heat, peptidases or inhibitors of
oxygen radicals and peroxides (25). The mechanism(s) mediating the phenomenon
thus remains unknown.
Finding that its DNA rapidly degrades in air suggested to us that R. cecicola may
be oxygen-intolerant because that gas triggers a mechanism which destroys its genetic
material. Other mechanisms mediating the organism's oxygen-intolerance cannot be
ruled out at this time. Whatever the mechanisms, however, the organism's sensitivity
to oxygen could have ecological significance. Oxygen may prevent such bacteria from
entering the habitat of the community of helical-shaped bacteria and spirochaetes
colonising the mucous gel nearest the epithelial membranes and in the crypts in the
caecum.
As noted earlier, the latter micro-organisms grow only in atmospheres containing
from 2 to 4% oxygen (29), which suggests that oxygen in small amounts passes from
the blood through the epithelial cells into the mucous environment where the helicalshaped organisms live. Oxygen in the region would be a powerful ecological factor
inhibiting oxygen-intolerant anaerobes from entering the habitat of the helical-shaped
organisms, even though the latter bacteria occupy the same mucous gel just below
the anaerobes (47). It follows from this hypothesis that the helical-shaped organisms
may serve as a barrier protecting the anaerobes in the mucus above them from the
oxygen. These exciting possibilities are under study.
*
* *
EL ECOSISTEMA DIGESTIVO Y SU COLONIZACIÓN. - D . C . Savage.
Resumen: El tracto gastrointestinal de los animales de la mayoría de las especies
es colonizado por una flora microbiana indígena constituida por micro­
organismos capaces de crecer anaeróbicamente. La mayoría de los organismos
son bacterias anaerobias que no toleran el aire y que colonizan el tracto
progresivamente en los recién nacidos, culminando en comunidades microbianas
en los adultos. Estas comunidades colonizan los habitats que, según las especies
animales, pueden situarse en el lumen, en la superficie epitelial o dentro de las
criptas de Lieberkühn, en cualquier parte del tracto. Una variedad de factores
determinan qué especies microbianas pueden formar comunidades en habitats
en animales de una determinada especie. Algunos de estos factores dependen
del animal huésped, de su dieta y de su medio ambiente y otros, de las diversas
270
especies microbianas que entran en interacción. Un factor importante es la
capacidad de los microorganismos para colonizar las superficies epiteliales.
Estamos estudiando algunas propiedades de los microorganismos capaces de
colonizar las superficies epiteliales gástricas o intestinales en ratas y ratones de
laboratorio y es probable que las propiedades en estudio hayan evolucionado
en los microorganismos para permitir a estos últimos adaptarse o colonizar sus
habitats superficiales. Nuestro estudio supone la aplicación de tecnología
molecular y genética para identificar macromoléculas específicas que resultan
importantes para permitir que los microorganismos permanezcan y llenen un
nicho (función) en las superficies epiteliales.
P A L A B R A S C L A V E : Epitelio intestinal - Flora microbiana indigena - Flora
microbiana n o r m a l - Roedores - Tracto gastrointestinal.
*
* *
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