# Interactions of the Intestinal Epithelium with the Pathogen and  the Indigenous Microbiota: A Three-Way Crosstalk

Authors: C. V. Srikanth; Beth A. McCormick
Journal: Interdisciplinary Perspectives on Infectious Diseases
 (2008)

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## Abstract

The mucosal surfaces of the gastrointestinal tract harbor a vast number of commensal microbiota that have coevolved with the host, and in addition display one of the most complex relationships with the host. This relationship affects several important aspects of the biology of the host including the synthesis of nutrients, protection against infection, and the development of the immune system. On the other hand, despite the existence of several lines of mucosal defense mechanisms, pathogenic organisms such as Shigella and Salmonella have evolved sophisticated virulence strategies for breaching these barriers. The constant challenge from these pathogens and the attempts by the host to counter them set up a dynamic equilibrium of cellular and molecular crosstalk. Even slight perturbations in this equilibrium may be detrimental to the host leading to severe bacterial infection or even autoimmune diseases like inflammatory bowel disease. Several experimental model systems, including germ-free mice and antibiotic-treated mice, have been used by various researchers to study this complex relationship. Although it is only the beginning, it promises to be an exciting era in the study of these host-microbe relationships.


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## Body

## 1. Introduction

A mature human gut harbors a vast number of bacterial residents referred to as
the commensal microflora or more recently as “microbiota.” It has been
estimated that this microbiota is made up of more than 1014 individual
bacteria comprising over 500 different species [1]. Notably, the composition of the
microbiota is individual specific and the type of species residing in the gastrointestinal
(GI) tract varies with the host organism’s age, diet, and health status [2]. In fact, the total number of microbes
in the human GI tract far exceeds (>10–100 times) the
sum of all our somatic and germ cells. The biological outcome of this vast and
complex population of microbes is that their genes (termed the microbiome)
synthesize about 100 times more proteins than the somatic cells of their host [3].Not
surprisingly, the human intestine is more densely populated with microorganisms
than any other organ and is a site where they exert a strong influence on human
biology. This is because the intestinal
mucosa serves as the primary border between the immune system and the external
environment, and in addition plays a central role in host-commensal flora
interactions. Accumulating evidence indicates that the gut microbiota is
instrumental in supporting energy metabolism and immune function of the host.
More recent studies suggest that the commensal microbiota play an important
role in the development of numerous conditions, including obesity [4, 5], diabetes [6], nonalcoholic fatty liver disease [7], inflammatory bowel disease [8], and perhaps cancer [9]. Unfortunately, the immense complexity
of gut flora together with its highly complicated interactions with intestinal
epithelium makes it a recalcitrant system to study. Although largely
unexplored, our gut microbiota plays an intricate and under-appreciated pivotal
role for our health and well-being. In this review we will discuss new
developments in the field that highlight the cellular and molecular basis of
the crosstalk between the host, the commensal microbiota, and pathogenic
bacteria in a healthy as well as a diseased GI tract.


## 2. Role of the Microbiota in the Gastrointestinal Tract

The
microflora of the intestinal microenvironment as a unit provides important
protective, metabolic, and trophic functions. Resident bacteria serve a central
line of resistance to colonization by exogenous microbes, and thus assist in
preventing the potential invasion of the intestinal mucosa by an incoming
pathogen. This protective function is known as the barrier effect or colonization
resistance and serves a number of important roles. For instance, adherent
nonpathogenic bacteria can often prevent attachment and subsequent entry of
suspected pathogens into epithelial cells, as well as compete for nutrient
availability. The commensal microbiota
also helps maintain GI nutrient homeostasis by administering and consuming all
resources. For example, dietary
nutrients are absorbed by the gut and together with various nonnutrient
compounds produced by the microbiota are cometabolize by host enzymes, such as
cytochrome P450 and conjugating enzymes in the liver [10]. The resulting metabolites that are
derived from both host and microbial processes are returned to the gut by the
bile for further metabolism or excretion [11]. This mutual and beneficial relationship
helps to dampen unwanted overproduction of nutrients, which could potentially
support intrusion of microbial competitors with a potential pathogenic outcome
for the host [12].Quite
remarkably, an absence of intestinal bacteria is associated with reduction in
mucosal cell turnover, vascularity, muscle wall thickness, motility, baseline
cytokine production, digestive enzyme activity, and defective cell-mediated
immunity [13]. Indeed, comparative studies in
germ-free and conventional animals have established that the intestinal
microflora is essential for the development and function of the mucosal immune
system during early life, a process that is now known to be important to
overall immunity in adults. For example, it has been well established that the
number of intraepithelial and lamina propria T cells is lower in germ-free
animals, a feature that is reversed upon the restoration of the normal flora [14]. Likewise, levels of secretory IgA are
low in the intestine of germ-free animals but are markedly increased upon
intestinal colonization of the commensal bacterium, Bacteroides thetaiotamicron [15]. Furthermore, the intimate relationship
between the commensal microbiota and the intestinal epithelium are involved in
shaping the memory mechanisms of systemic immunity, such as oral tolerance.
This was initially recognized by the discovery that the systemic response to a
specific pathogen can be abrogated after ingesting the antigen; this effect
continues for several months in conventionally colonized mice, whereas in
germ-free mice systemic unresponsiveness persists for only a few days [16]. Therefore, the innate immune system
discriminates between potential pathogens from the commensal microbiota by
inducing tolerance to microbial epitopes. This, in turn, dampens responses to
commonly encountered foodstuffs and other environmental antigens. Collectively,
these examples help to illustrate the important concept that the commensal
microbiota profoundly influence the development of the gut mucosal immune
system and are essential in preventing exogenous pathogen intrusion.The intestinal microflora also makes
important metabolic contributions by producing vitamin K, folate, and short-chain
fatty acids (a major energy source for enterocytes), and mediates the breakdown
of dietary carcinogens as well [2, 17]. Perhaps the major metabolic function of the colonic
microflora is the fermentation of nondigestible carbohydrates. These
nondigestible carbohydrates include large polysaccharides (i.e., resistant
starches, pectins, cellulose), some oligosaccharides that escape digestion, as
well as unabsorbed sugars and alcohols. The primary metabolic endpoint of such
fermentation is the generation of short-chain fatty acids (acetate,
proprionate, butyrate). A fundamental role of short-chain fatty acids on
colonic physiology is their trophic effect on the intestinal epithelium.
Therefore, short-chain fatty acids appear to play an essential role in the
control of epithelial cell proliferation and differentiation in the colon.
Recent studies have also shown effects of butyrate on intestinal barrier
function [18].
Moreover, it has been shown that commensal bacterial can modulate gene
expression in the host in order to create a sustainable environment for
themselves, while at the same time prevent the growth of other competitive
bacteria within the intestinal ecosystem [15].For the host to thrive and produce more
gut residents, the gut microbial ecosystem must be functionally stable over
time despite the internal dynamics of the community. Constituent bacteria are
expected to have a high degree of functional redundancy between species, so
that the loss of one lineage does not adversely impact the homeostatic balance
of the intestinal microenvironment [19]. While it is unclear how the selective
pressures, microbial community dynamics, and the intestinal microenvironment
shape the genome and subsequent functions of members of the gut microbiota,
there are some exciting new developments in the field. For example, Gordon et
al. have introduced the provocative concept that the evolution of the gut
microbiome also likely plays a significant role in shaping the evolution of
humans [19]. This tenet is founded on experiments in
which this team of investigators sequenced the genomes of two gut-dwelling
Bacteroidetes and compared their genomes to the genomes of other bacteria that
live both inside and outside of the human body. Quite remarkably, they
discovered that lateral gene transfer, mobile genetic elements, and gene
amplification play an important role in affecting the ability of the
Bacteroidetes to vary their cell surface, sense their environment, and harvest
nutrient resources present in the distal intestine [19]. Importantly, these findings lay the
conceptual groundwork to suggest that adaptation to the gut ecosystem is a
dynamic process that includes acquisition of genes from other microorganisms,
and further underscores the significance of considering the evolution humans
from the perspective of the evolution of the microbiome [19, 20].


## 3. Restricting Pathogens and Commensal from Invading beyond the Mucosal Surface

The
host is protected from potentially harmful enteric microorganisms by the
physical and chemical barriers created by the intestinal epithelium that are
primarily comprised of absorptive villus enterocytes [21]. The apical surface of the enterocytes
are highly differentiated structures consisting of rigid, closely packed
microvilli whose membranes contain stalked glycoprotein enzymes [22, 23]. In addition, the tips of enterocyte microvilli are
coated with a 400–500 nm thick
meshwork referred to as the filamentous brush border glycocalyx [24] and is composed of highly glycosylated
transmembrane mucins [25, 26]. The intestinal epithelial barrier is also composed of
enteroendocrine cells, goblet cells, and Paneth cells. Microfold (M) cells are
also present in the follicle-associated epithelia where they represent a
morphologically distinct epithelial cell type whose primary function
is in the transport of macromolecules, particles, and microorganisms
from the lumen to underlying lymphoid tissue [27, 28]. Intercellular junctional complexes that are composed of
tight junctions, adherens junctions, and desomosomes maintain the integrity of
the epithelial barrier. The most apical components of the junctional complex
are the epithelial tight junctions, which are highly regulated and serve to
create a semipermeable diffusion barrier between individual cells (Figure 1(a)).
Collectively, these features facilitate the intestinal epithelium to act as a
physical barrier to prevent unwanted bacteria from gaining access to the host.(a) Healthy epithelial
surface. A healthy intestinal epithelial surface acts as a physical and biochemical
barrier with key features including the
apical brush border, the mucus layer, the presence of antimicrobial peptides
(blue black dots) in the lumen, the glycocalyx, and the epithelial tight
junctions. Also seen in the illustration are numerous commensal bacteria and a
dendritic cell sampling the lumen with its extended dendrites (yellow). (b) Key features ofS. typhimurium infected epithelium. Such
host pathogen interactions involve translocation of bacterial effectors (green
circles) into the epithelial cells, membrane ruffling, bacterial endocytosis,
and SCV formation. Chemoatractants are secreted by the epithelial surface that
leads to PMN influx. SCV: Salmonella containing vacuole. (c) Intestinal epithelial
surface of an antibiotic-treated patient showing enrichment of a set of
antibiotic resistant members of the commensal microflora (light blue and brown)
such as C. difficle and E. faecalis. The C. difficle proteins, TcdA and Tcdb (red circles) act
intracellularly as glycosyltransferases and inhibit Rho, Rac, and Cdc42. The
effect of these modifications lead to actin condensation, transcriptional
activation of several genes and apoptosis. Other mechanisms that are triggered
include basolateral IL8 secretion, apical Hepoxillin A synthesis, and PMN
influx in the apical surface.
(a)(b)(c)The intestinal epithelium also
provides a unique surface that is armed with a bounty of specialized cells that
produce mucus, antimicrobial peptides, and antimicrobial molecules, which
together form the front line of defense against pathogenic microorganisms (Figure1(a)). The mucus layer is secreted by the goblet cells and this layer overlies
the intestinal epithelium to create a physical blockade against offending
enteric microbial pathogens. For example, it has been demonstrated that
secreted mucus acts as a barrier to Yersinia enterocolitica [29], rhesus rotavirus [30], and Shigella
flexneri [31]. The commensal microbiota has also been
found to regulate the production of intestinal mucins, which consequently
inhibits the adherence of numerous pathogenic bacteria to intestinal epithelial
cells [32–34]. Paneth cells are
another important cell type that are involved in intestinal defense against
potential harmful pathogenic bacteria. These cells are present at the base of
the crypt of Lieberkühn [35] and have been shown to produce a number
of antimicrobial peptides. In addition, the gastrointestinal expression of
antimicrobial peptides is evolutionarily conserved [36], and to date, α-defensins (HD), β-defensins (hBD), and cathelicidins have been
identified in humans [37]. Paneth cells also produce a number of
antimicrobial molecules, including lysozyme, phospholipase A2, and
angiogenin-4 (reviewed in [37]). Therefore, it is inferred by numerous
studies that Paneth cells are able to control the bacterial ecosystem (Table
1).Table 1
Antimicrobial peptides/proteins and their targets.
ClassExamplesExpressionActionReferencesα-defensinHD-5,
HD-6Paneth cellsL. monocytogenes[38–41]E. coliS. typhimuriumβ-defensinhBD-1IECsP. aeruginosa[42–46]hBD-2E. coliCandida
albicansCathelicidinhLL37IECsSalmonella[47, 48]AngiogeninAngiogenin-4Paneth cellsGram positive[49–51]BacteriaC-type lectinRegIIIγIECsGram positive[52–54]BacteriaAngiogenin-4 is expressed mainly in the
small intestine, cecum, and colon and acts on Gram-positive bacteria [49, 50]. However, most antimicrobial peptides expressed by
mammalian epithelial cells are members of peptide families that mediate
nonoxidative microbial cell killing by phagocytes [50]. These amphipathic molecules interact with and lyse bacterial membranes [55]. Defensins
generally possess a broad range of antimicrobial activity (Table 1). In
particular, human intestinal defensin-5 has been shown to kill Listeria monocytogenes, E. coli, and Candida albicans [40]. Additional evidence supporting a
critical role for defensins in vivo
was demonstrated in a study utilizing human defensin-5 transgenic mice; these
mice exhibited marked resistance to oral challenge with virulent Salmonella enterica serovar Typhimurium (S. typhimurium) [39]. The
intestinal epithelial cells also express another class of antimicrobial
peptide, the cathelicidins (LL-37/Cap18), in which a cathelin domain is linked
to a peptide with antimicrobial activity [56]. LL-37 is expressed within the
epithelial cells located at the surface and upper crypts of normal human colon.
Although little or no expression is seen within the deeper colonic crypts or
within epithelial cells of the small intestine, studies in mice have determined
these molecules to be protective against bacterial pathogens [47]. Interestingly, the expression of these
factors, unlike the angiogenins, is not induced by the presence of pathogenic
bacteria but rather their secretion is triggered by the commensal microbiota
and/or their derivatives. A recent addition to this growing list of intestinal
antimicrobial includes RegIIIγ, which has been shown to be toxic to
Gram-positive bacteria [52]. RegIIIγ is a C-type lectin that binds to the
carbohydrate moiety of bacterial cell wall constituent, petidoglycan. Recent
studies have further shown that the expression of RegIIIγ is strongly dependent upon the presence of the
gut microflora since in germ-free mice RegIIIγ expression is severely repressed [53] (Table 1).The intestinal epithelium also provides a
surface where the host can sense the microbial microenvironment in order to
elicit an appropriate defense response by releasing an array of signaling
molecules (i.e., chemokines and cytokines). These molecules then trigger the recruitment of
leukocytes to initiate an early inflammatory response. Paradoxically, however,
although continuously exposed to Gram-positive and Gram-negative bacteria and
their products (i.e., lipopolysaccarhide (LPS), peptidoglycan, and lipoprotein)
the normal healthy intestinal mucosa maintains a mechanism of
hyporesponsiveness to the lumenal microbiota and their products. Exaggerated
inflammatory responses in the absence of pathogenic bacteria would be otherwise
deleterious [57, 58]. Accordingly, the normal intestinal epithelial host
defenses are able to accurately interpret the complex microbial environment in
order to discriminate between permanently established commensal microbes and
episodic pathogens.At the core of this strategy the
endogenous microbiota all share “self” signature molecules termed
microbe-associated molecular patterns [59]. However, upon infection of a pathogenic
organism, the host immune response is activated by the specific recognition of
“nonself” molecular structures known as pathogen-associated molecular patterns.
The epithelial cells are able to sense the microenvironment within the gut by
means of pattern recognition receptors (PRRs) that include Toll-like receptors
(TLRs) and nucleotide-binding oligimerization domain (NOD) proteins [38, 60–63]. TLRs are evolutionary conserved and are characterized by
an extracellular leucine rich repeat (LRR) domain (involved in ligand
recognition), as well as an intracellular Toll/interleukin-1 receptor-like
domain (involved in proinflammatory signal transduction) [60, 64–66]. In addition, two NOD proteins (NOD1 and NOD2) function as
intracellular sensors of bacterial products in the induction of an inflammatory
response [60, 64–67].These
PRRs recognize bacterial factors, such as LPS, lipoproteins, flagellin,
unmethylated-CpG DNA, and a large number of other specific components.
Regulation of the expression and the specific location of TLRs and NODs in
intestinal epithelial cells fosters efficient immune recognition of the
commensal microflora and maintains a delicate balance; permitting a basal level
of signaling events to proceed, while at the same time restraining innate
immune responses. For instance in a healthy intestine, epithelial cells express
very little or no TLR2, TLR4, and CD14, and as a result minimizes the
recognition of commensal LPS [68, 69]. TLR5, which recognizes bacterial flagellin, has been
reported to be expressed exclusively on the basolateral surfaces of the
epithelial cells. This TLR is ideally positioned to detect its ligand,
translocated flagellin [70]. Moreover TLR3, TLR7, TLR8, and TLR9 are
expressed in the intracellular endosomal compartments [71]. These intracellular PRRs would not
ordinarily encounter luminal commensal bacteria or those attached to the apical
surface of intestinal epithelial cells but are well positioned to recognize
pathogenic bacteria that actively breach the epithelial barrier. As an
additional measure, commensal bacteria have the ability to induce the
expression of intestinal alkaline phosphatase, which not only dephosphorylates
dietary lipids but also dephosphorylates the LPS of commensal flora resulting
in reduced toxicity in mammals [72].Nonpathogenic microorganisms may also be
able to selectively attenuate the NF-kB pathway as mechanism of intestinal
immune tolerance. Neish et al. initially reported that colonization of a human
model intestinal epithelium with certain strains of nonpathogenic bacteria
could dampen the host cell responses to subsequent proinflammatory challenges
by blocking the proinflammatory/antiapoptic NF-κB pathway [73]. This effect is mediated by the inhibition of IκB-α ubiquitination, which prevents regulated
IκB-α degradation, NF-κB nuclear translocation, and subsequent
activation of proinflammatory/antiapoptic genes. IκB-α ubiquitination is catalyzed by E3-SCFβ−TrCP ubiquitin ligase [74], which is regulated via covalent modification of the
cullin-1 subunit by the ubiquitin-like protein NEDD8 [75, 76]. Recently, it was determined that the interaction of
nonpathogenic bacteria with epithelial cells results in the rapid loss of
neddylated Cul-1 and consequent repression of the NF-κB pathway [77]. Collectively, this set of observations
underscores the ability of intestinal bacterial communities to influence
eukaryotic processes, and perhaps more specifically demonstrates inflammatory
tolerance of the mammalian intestinal epithelia.


## 4. How Pathogens Overcome the Epithelial Barrier

As
described above, the intestinal epithelium has evolved a rather formidable
fortress to guard against microbial invasion. However, through a process of
coevolution, potential harmful enteric microorganisms have evolved counter
strategies to hijack the cellular molecules and signaling pathways of the host
to become potentially pathogenic. As an initial step in the infection process,
certain enteric pathogens target specific epithelial cell structures, including
glycoproteins and glycolipids, which serve as receptors for bacterial
attachment [78]; thus, enabling them to exploit the
underlying signal transduction pathway. Other strategies utilized by invading
enteric pathogens, such as S.typhimurium and Shigella flexneri have evolved a sophisticated strategythat
directs the entry of the enteric pathogen into intestinal epithelial cells. This process requires the expression of a bacterial type III protein secretion system (TTSS), the function of which is to deliver a set of effector proteins into the host cell [79–81]. These effector proteins co-opt host cell signal
transduction cascades as a clever means of subverting normal host cell
processes by triggering a marked rearrangement of the host
cytoskeleton. This entry mechanism termed bacterial mediated endocytosis drives
bacterial entry and facilitates the pathogen to cross the epithelial barrier as
well as to induce a proinflammatory response [79–81].The
latter step in this process can be achieved by direct cytotoxic injury,
intracellular migration, disruption of the epithelial tight junctions, or
indirectly by inducing neutrophil infiltration. Although several bacterial
pathogens have been able to modulate epithelial tight junctions to
their own advantage, the direct interaction of a bacterial virulence
factor on component proteins of the tight junction has been proposed
only in a few instances [82]. It is well documented that anumber of enteric pathogens perturb the intestinal epithelial
barrier and impact TER or paracellular permeability, most often with
an alteration in the arrangement of tight junctional component
proteins by mechanisms that are unique for different pathogens [82]. For example, Clostridium difficile toxins A and B enhance epithelial
cell permeability by disrupting actin microfilaments within the
perijunctional ring [83], and enteropathogenic Escherichia coli disrupt the epithelial
barrier by the phosphorylation of myosin light chains [84]. With respect to S. typhimurium, in vitro models of infection have
revealed an alteration of epithelial permeability and loss of
barrier function, which involves rapid changes in both tight junction
permeability and transcellular conductance [85, 86]. Recent studies further indicate that the Salmonella effector protein SigD (also called SopB), which is encoded in Salmonella pathogenicity island-1 (SPI-1), is able to elicit a reduction in
epithelial barrier function, perhaps via activation of PKC [87]. Also, the effector proteins SopB, SopE,
SopE2, and SipA are necessary to disrupt the epithelial barrier and
alter the distribution of at least some tight junction proteins [88, 89]. Such
perturbations in the components of the tight junction lead to enhanced
bacterial translocation
and infiltration of neutrophils across the intestinal barrier. Therefore, the
ability to regulate the molecular composition of the tight junctions
facilitates the pathogenecity of S.
typhimurium by fostering its uptake and distribution within the host (Figure 1(b)) [85].S. flexneri has a
distinct mode of pathogenesis that involves entry into colonic epithelial
cells from the basolateral surface [90], thereby requiring its
relocation from the lumenal to the underlying surface of the
epithelium. This translocation event has historically been
attributed to the uptake and transport by M cells [91]. However, it has since been established that Shigellae are also capable of altering components of the
tight junctional complex, allowing the bacteria to traverse the
paracellular space to reach the basolateral surface; an event that
also decreases barrier function [92]. Once at the basolateral surface, Shigellae rapidly invade and
disseminate through the epithelium, causing a further decrease in
barrier function [92–94] through the action of a TTSS system and
additional proteins encoded on a large virulence plasmid [94–97].Enteric
pathogens cause a variety of diseases in humans but one undeniable symptom is
the presentation of gastroenteritis. Some bacterial enteric infections are
characterized by disruption of the normal movement of electrolytes and water
across the epithelium, which is converted from a state of net fluid absorption
to one of net fluid secretion [98]. Secretory diarrhea, as a result of
epithelial chloride secretion, has long been regarded as a host defense
mechanism. This is based on the notion that increased fluid and electrolyte
movement into the gut lumen helps to inhibit adherence of pathogenic organisms
by “flushing” them from the body. However, it could also be argued that the
induction of pathogen-induced diarrhea is a way to ensure transmission to new
hosts, and thus pathogenic fitness [99]. These ideas are not mutually exclusive
and secretory diarrhea may be advantageous to both host and pathogen.Pathogenic bacteria cause diarrhea by
multiple mechanisms.Vibrio cholerae reside in the lumen of the small intestine and produce toxins, which alter ion
absorption and/or secretion [100, 101]. Other bacteria such as Shigella and enteroinvasive E. coli invade and
destroy the colonic epithelium leading to dysentery [102]. More recently pathogenic E. coli have been shown to increase
chloride ion secretion from intestinal epithelia by upregulating the expression
of the receptor for the neuropeptide galanin 1 [103]. Rotavirus, another important cause of
diarrhea in infants, induces this condition by activating the enteric nervous
system [104, 105].A large influx of neutrophils (PMNs) into
the mucosa and lumen from the underlying vasculature is a significant feature
of intestinal bacterial infections [105, 106]. During infection of epithelial cells by enteric pathogens
such as S. typhimurium and S. flexneri, IL-8 is synthesized and
secreted baslaterally. Such basolateral IL-8 release imprints subepithelial matrices
with long-lived haptotactic gradients that serve to guide neutrophils through
the lamina propria to a subepithelial position [107]. However, basolateral IL-8 release is
insufficient to induce the migration of neutrophils across the intestinal
epithelium, suggesting that the production of other inflammatory mediators,
whose release would probably be polarized apically, is important for the
execution of this step in the inflammatory pathway [107, 108]. In support of this contention, Kucharzik et al. recently developed a
double transgenic mouse model with the ability to induce human IL-8 expression
restricted to the intestinal epithelium [109]. The results from this transgenic model showed that
although acute induction of IL-8 in the intestinal epithelium is sufficient to
trigger neutrophil recruitment to the lamina propria, additional signals are
required for neutrophil transepithelial migration and mucosal tissue injury.
Indeed, recent evidence suggests that the eicosanoid, hepoxilin A3,
is secreted apically and is responsible for the final step of neutrophil
transepithelial migration into the gut lumen [110, 111]. This process is
quite complex as distinct signaling pathways mediate S. typhimurium invasion, induction of CXCL8 secretion, and
induction of hepoxilin A3 secretion [111–113].The ability ofSalmonella serotypes to elicit PMN transmigration in vitro correlates with their
ability to cause diffuse enteritis (defined histologically as transepithelial
migration of neutrophils), but not typhoid fever in humans [114]. Moreover, large-scale PMN transepithelial
migration causes decreased barrier function [115]. Studies exploring the mechanism underlying the release of HXA3 during
infection with S. typhimurium revealed the involvement of the S. typhimurium type III secreted effector protein, SipA [116]. The S.
typhimurium effector protein, SipA, promotes a lipid signal transduction cascade
that recruits an ADP-ribosylation factor 6 guanine nucleotide exchange factor
(such as ARNO) to the apical plasma membrane. ARNO facilitates ADP-ribosylation
factor 6 activation at the apical membrane, which in turn, stimulates
phospholipase D recruitment to and activity at this site. The phospholipase D
product, phosphatidic acid, is metabolized by a phosphohydrolase into
diacylglycerol, which recruits cytosolic protein kinase C (PKC)-alpha to
the apical membrane. Through a process that is less understood, activated
PKC-alpha phosphorylates downstream targets that are responsible for the
production and apical release of HXA3, which drives transepithelial
neutrophil movement [117].


## 5. Prototypical Interactions between Pathogenic Bacteria and Commensal Microbiota

There
are ample lines of evidence to support the emerging concept that a change in
the composition of the commensal microbiota alters the intestinal
microenvironment making this niche vulnerable to pathogenic insult. In this
section we discuss examples to illustrate the remarkable crosstalk between the
host, its intestinal microbiota, and potential pathogenic bacteria.It
has been well documented thatS.
typhimurium causes a systemic (typhoid fever) infection in mice while in
humans this enteric pathogen causes gastroenteritis. However, Barthel et al. discovered
that pretreatment of C57BL/6 mice with streptomycin, an antibiotic that kills
facultative anaerobes, followed by infection with a streptomycin-resistant
strain of S.typhimurium produced a robust intestinal inflammatory response [118]. Such enteritis is primarily characterized by inflammation
in the cecum, and also presents with several of the typical pathological
hallmarks of acute Salmonella-induced
gastroenteritis in humans, including PMN infiltration and epithelial cell
erosion. This is an intriguing result since the only difference between the
untreated and streptomycin treated mice is the alteration of the commensal
flora; thus, demonstrating that the presence of the microflora plays a
protective role against pathogenic invaders. This study also substantiates the
long-standing finding of Barrow and Tucker who found that pretreatment of a
chicken’s cecum with three different strains of E. coli significantly reduced infection with Salmonella as compared to untreated animals [119]. Additionally, Hudault et al. (2001)
determined that the presence of a single species of E. coli in the gut could restrict the infection of S . typhimurium as compared to its germ-free
counterpart [120].More
recently, Stecher et al. used theS.
typhimurium colitis model to investigate competition between an enteric
pathogen and the host microbiota [121]. This group found that inflammatory responses induced by S. typhimurium led to profound
perturbations in the composition of the commensal microbiota as determined by
16S rRNA. The inflammatory host responses induced by S. typhimurium not only changed the microbiota composition but also
suppressed its growth, thereby, overcoming colonization resistance. In contrast,
an avirulent Salmonella mutant
defective in triggering inflammation was unable to overcome colonization
resistance. These results raise an interesting point in that perhaps the
intestinal inflammation induced by S.
typhimurium might be a crucial event in order to overcome colonization
resistance. In this respect, triggering the host’s immune defense may shift the
balance between the protective microbiota and the pathogen to favor the
pathogen. The idea that the intestinal microbiota can be altered by invading
pathogens is further supported by Lupp et al. who found that host-mediated
inflammation in response to an infectious agent induced alterations in the
colonic community that not only resulted in the elimination of a subset of
indigenous microbiota but also led to the growth of the Enterobacteriaceae
family [122]. Moreover, in children undergoing
treatment for diarrhea, fluctuations in the intestinal microflora were observed
for both rotaviral and nonrotaviral-induced diarrhea [123]. This phenotype was reversed and the
normal microflora was re-established after about three months of the disease
episode. Other studies have investigated the role of the intestinal microbiota
during infectious disease transmission. In particular, Lawley et al. describe a
model in which persistently infected 129X1/SvJ mice provide a natural model of
transmission. In this model, only a subset of mice termed “supershedders” could
shed high levels of bacteria in their feces. Whereas immunosuppression of the
infected mice did not induce the supershedder phenotype, antibiotic treated mice
displayed a high supershedder phenotype [124]. Together, these studies suggest that
the intestinal microbiota plays a critical role in controlling pathogen
infection, disease, and even transmissibility.There
are also examples in which members of the commensal microflora are able to
cause disease. This is specifically illustrated byEnterococcus faecalis, a prominent member of the GI tract
microbiota. In a healthy intestine these bacteria behave as a normal resident of
the intestinal ecosystem. However, in individuals undergoing antibiotic
treatment or those who are immunocompromised, E. faecalis is able to colonize new niches of the intestinal
microenvironment as a certain subgroup of this species is antibiotic resistant
(Figure 1(c)). Under such compromised conditions, E. faecalis can infect and spread to other sites of the host such
as the bloodstream, urinary tract, and surgical wounds. Not surprisingly, the
subgroup population harboring the antibiotic resistance genes also has genetic
elements conferring infectivity and virulence. Furthermore, the genome sequence
of E. faecalis strain V583, the most
causative agent of vancomycin resistant enterococcal infection in America, [125] was recently reported [126]. Recent studies have determined that more than 25% of the E. faecalis genome is most likely
derived from mobile or foreign DNA, which might have contributed to the rapid
acquisition and dissemination of drug resistant strains [126]. Another example is illustrated by Clostridiumdifficle, a
Gram-positive bacterium that can harmlessly inhabit the human intestine.
However, certain individuals undergoing antibiotic therapy, as a result of
their altered intestinal microflora, presented with C. difficle infection accompanied with severe intestinal colitis
(Figure 1(c)) [127].Commensal
bacteria, such asBacteroides fragilis,
may also inhibit other opportunistic members of the intestinal microflora from
causing disease [128]. B.
fragilis is a Gram-negative bacterium that resides in a healthy human
intestine. Normally, this bacterium expresses a surface carbohydrate capsule
known as polysaccharide A (PSA), which contributes to many beneficial
activities underlying the immune development of the host, including activation
of CD4+ T cells, and stimulation of the innate immune responses through TLR2
signaling. Mazmanian et al. determined that B.
fragilis protects the host from Helicobacter
hepaticus-induced colitis in experimental mice. However, in animals
harboring B. fragilis strains that do
not express PSA, H. hepaticus colonization led to disease and production of proinflammatory cytokines induced
by intestinal immune cells [128, 129]. Thus, in healthy individuals it appears that PSA from B. fragilis is necessary to confer some
beneficial activity. In spite of this, PSA was also found to potentiate the
ability of B. fragilis to cause
disease in patients who have a compromised mucosal surface, such as
postsurgical patients. This function is initiated upon submucosal entry of the
bacteria during which PSA activates CD4+ T cells leading to abscess formation [130].


## 6. Role of Bacteria in Inflammatory Bowel Disease

Recent evidence from a variety of investigative avenues
implicates abnormal host-microbial interactions in the pathogenesis of
inflammatory bowel disease (IBD). In fact, IBDs preferentially occur in the
colon and distal ileum (i.e., locations that contain the highest concentrations
of intestinal bacteria). An important role for microbial agents in the
pathogenesis of IBD is inferred by numerous recent studies, which conclude the
bacterial flora differs between patients with inflammatory bowel disease (IBD)
and healthy individuals. Moreover, accumulating evidence suggests that the
composition and function of the microbiota in patients suffering with IBD are abnormal.Ninety-nine
percent of the gut microbiota in healthy individuals is composed of species
within four bacterial divisions:Firmicutes, Bacteroidetes, Proeobacteia, and Actinobacteria [131, 132]. Investigation of the microbial diversity in active IBD is
a highly pursued topic of interest and is an area of research still at its
infancy. In IBD patients, early returns have suggested that there is a decrease
in the number of beneficial bacteria, such as Bifidobacterium and Lactobacillus spp., and an increase in pathogenic bacteria, such as a Bacteroides and Escherichiacoli [132–136]. Such dysbiosis induces a breakdown in the balance between
putative spp. of protective versus harmful bacteria, and may promote
inflammation. Other studies have shown that there is a decrease in microbial
diversity that accompanies the increased numbers of Enterobacteriaceae,
including E.coli, with decreased numbers of Firmicutes,
and a particular decrease in Clostridium species. As convincing as this data is, there is still a lack of evidence to
denote whether a specific pathogen is responsible for onsets or relapses of IBD
[132]. Further, the most compelling studies
are derived from animal models. Regardless, a number of organisms have been
implicated in Crohn’s disease, with Mycobacterium
paratuberculosis and E. coli drawing a great deal of attention [137].Patients
with IBD have higher numbers of mucosa-associated bacteria than control patients
[138], and the generalized or local dysbiosis
observed is due to the presence of low numbers of normal bacteria and high
numbers of unusual bacteria with a decrease in biodiversity. The composition of
the increased numbers of bacteria attached to the intestinal epithelium of IBD
patients are from diverse genera. Bacteroides
spp., in particular, has been identified as a predominate member of the
epithelial layer, and in some instances was located intracellularly [136]. While this remains an intriguing observation, the role of Bacteroides in IBD is still unclear.
Furthermore, distinct adherent or invasive E.
coli has been identified in the ileal mucus of patients with Crohn’s
disease, and the involvement of a new potentially pathogenic group of adherent
invasive E. coli (AIEC) has been
suggested [139]. For instance, in studies aimed to
assess the predominance of E. coli strains associated with the ileal mucosa of Crohn’s disease patients, E. coli was recovered from 65% of
chronic lesions and from 100% of the biopsies of early lesions. By comparison,
3–6% of the E. coli was recovered
form healthy ileal mucosa. E. coli was also abnormally present (50–100% of the total
number of aerobes and anaerobes) in early and chronic ileal lesions of CD
patients [140, 141]. These observations were confirmed in a subsequent study in
which adherent E. coli was found in
38% of patients with active ileal Crohn’s disease [133]. This study also revealed that the
number of E. coli in situ correlated with the severity of the disease, and that the invasive E. coli was also restricted to the
inflamed mucosa. Interestingly, the recovered E. coli strains were predominantly novel in phylogeny, displayed pathogen-like
behavior in vitro, and expressed virulence factors [133].It
is suspected that the abnormal colonization of the lieal mucosa is largely due
to increased expression of CEACAM6, a receptor for adherent-invasiveE. coli [142]. However, Crohn’s disease patients also exhibit defective
microbial killing mechanisms that result in increased exposure to commensal
bacteria. For example, Crohn’s disease patients have defective antimicrobial
peptide production, including α-defensin 5 in ileal disease and human β-defensin 2 in Crohn’s colitis [143, 144]. This is accompanied by functional abnormalities in the
killing of Bacteroides vulgatus, E. coli, and Enterococcus
faecalis [145]. In addition, NOD2 polymorphisms in
Crohn’s disease are associated with selective decrease in α-defensin production by Paneth cells, as well
as in defective clearance of intracellular pathogens by colonic epithelial
cells [146]. Thus, combined with defective
antimicrobial peptide function in Crohn’s disease the functional changes
described above provide a reasonable rationale for the profound increase in
mucosally associated Enterobacteriaceae. Also, in light of the alteration in
the composition of the luminal microbiota, it is perhaps not surprising that
Crohn’s disease has features that might be the consequence of a microbial process.
This is exemplified by the noted infection of Peyer’s patches and lymphoid
aggregates, and the presence of ulcerations, microabscesses, fissures, fistulas, granulomas,
and lymphangitis [137].As evidence accumulates to suggest that
dysbiosis in IBD patients induces a breakdown in the balance between putative
spp. of protective versus harmful bacteria, one potential new method of
intervention lies in the modulation of the enteric flora. Indeed, current
studies suggest that probiotics might offer an alternative or adjuvant approach
to conventional IBD therapies by altering the intestinal microflora and, in
turn, modulating the host immune system. Probiotics are defined as living food
supplements or components of bacteria that have a beneficial effect on human
health. Indeed, probiotic activity has been associated withLactobacillus, Bifidobacteria, Streptococcus, Enterococcus, nonpathogenic E. coli, and Saccharomyces bourlardii [147, 148].Probiotic
supplements may balance the indigenous microflora in IBD patients. A growing
body of literature supports this emerging concept, which suggests that
probiotics have therapeutic effects in ulcerative colitis, Crohn’s disease and
pouchitis [147, 148]. The rationale for employing probiotics in the treatment of
IBD is underscored by the proposed pathogenic role of the intestinal microflora
in this disease. Numerous studies support the notion that introduction of
probiotics to the GI tract can alter the enteric microflora in IBD patients,
which in turn has a profound effect on intestinal defense mechanisms, including
(i) inhibiting microbial pathogenic growth, (ii) increasing epithelial cell tight
junctions and permeability, (iii) modulating the immune response of the
intestinal mucosa, (iv) increasing the secretion of antimicrobial products, and
(iv) eliminating pathogenic antigens [149–151]. Thus, such broad mechanistic effects of probiotics may
explain the beneficial effects observed.Probiotic
preparations are primarily based on a variety of lactic acid bacteria
(lactobacilli, bifidobacteria, and streptococci), which under healthy
conditions are normal and important components of the commensal microbiota. In
addition, probiotic mixtures often contain some nonpathogenic bacteria that
includeE. coli, enterococci, or
yeast (Saccharomyces bourlardii) [152]. Probiotic strains also need to satisfy
important criteria. First, probiotics must be safe and tested for human use [149, 152]. In addition, such strains should be of human origin,
resistant to acid and bile, and survive and be metabolically active within the
intestinal lumen. Probiotics must also be antagonistic against pathogenic
bacteria as they produce antimicrobial substances, compete within the GI tract,
and promote a reduction in colonic pH.Many
clinical trials have documented that probiotics can achieve and maintain
remission in patients with ulcerative colitis, and also prevent and maintain
remission of pouchitis. However,
probiotics seem to be ineffective in Crohn’s disease [153]. Although controlled clinical trials are
still required to investigate the unresolved issues related to efficacy, dose,
duration of use, single or multistrain formulation, and simultaneous use of
probiotics, synbiotics, or antibiotics, the preliminary data for the
therapeutic use of probiotics in selective patients with mild to moderate IBD
are encouraging.



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