Mecanismos Celulares de La Diarrea

7/28/2019 Mecanismos Celulares de La Diarrea

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Introduction

At its core, diarrhea is simply an altered movement o ions andwater that ollows an osmotic gradient. Under normal conditions,the gastro-intestinal tract has tremendous capacity to absorb uidand electrolytes, where 89 liters o uid are presented to the intes-tine daily and only 100200 ml are excreted in the stool. Enteric

pathogens, however, can alter this balance towards net secretion,leading to diarrheal disease. The altered movement o ions canoccur either through transporters or the lateral spaces betweencells, which are regulated by tight junctions (Fig. 1). In thisregard, some transporters seem to be tightly coupled with water

Correspondence to: Kim Hodges; Email: [email protected] / Ravinder Gill;

Email: [email protected]

Submitted: 10/14/09; Revised: 12/15/09; Accepted: 12/28/09

Previously published online:

www.landesbioscience.com/journals/gutmicrobes/article/11036

reviewreview

movement, including sodium-dependent glucose transporter(SGLT1), Na+/H+ exchanger isoorm 3 (NHE3) and the apicalCl-/HCO

3- exchanger, downregulated in adenoma (DRA). The

classical secretory diarrhea caused by cholera toxin (CT) is due tocAMP-dependent activation o the cystic fbrosis transmembraneconductance regulator (CFTR), a Cl- channel (Fig. 1). A lternately,changes in Ca2+ levels increase the activity o the calcium activated

chloride channel (CLCA). In some cases, as or CT, the increasein Cl- secretion is paired with a decrease in Na+ absorption. Inaddition the direct reduction o water transport proteins, such asaquaporins, results in less uid absorption (Fig. 1).

Enteric pathogens can either directly modulate epithelialion transport processes and barrier unction or do so indirectlythrough inammation, neuropeptides or loss o absorptive sur-ace. For example, pathogens such as the intestinal parasiteGiardia cause loss o brush border absorptive surace and diuseshortening o villi. Similarly, enteropathogenic E. coli (EPEC)cause eacement o microvilli, which decreases the surace areaor nutrient absorption and causes increased osmolarity o the

intestinal contents and malabsorption. However, recent evidencesuggests that the rapid onset o diarrhea induced by EPEC couldresult rom direct eects on intestinal epithelial ion transportprocesses. Several invasive pathogens, including Shigella andSalmonella species, cause an inammatory diarrhea character-ized by ever and polymorphonucleocytes (PMNs) in the stool.PMNs regulate absorption through cytokine secretion but alsohave a more direct role through the secretion o a precursor toadenosine, a secretagogue that activates CFTR. C. dicleandrotavius inection also work indirectly through modulation oion transport subsequent to cytokine secretion and activation oenteric nerves via neuropeptides.

This review highlights selected pathogens, which provide

both a broad overview o the mechanisms pertaining to iontransport and barrier unction that underlie pathophysiology odiarrhea, and presents recent advances regarding specifc inec-tious agents. Major emphasis will be placed on the mechanismsunderlying the pathogenesis o bacterial diarrhea, which has beenextensively studied in recent years and has served as an impor-tant prototype or understanding regulation o intestinal epithe-lial processes at the cellular and molecular level. However, themechanisms underlying recent advances in viral and parasiticdiarrhea will also be discussed. An increased understanding othese regulatory mechanisms is important to completely defne

infctous dahaCellular and molecular mechanisms

Km Hodgs and rand Gll

Univeristy of Illinois at Chicago; Digestive Disease and Nutrition; Chicago, IL USA

Both authors contributed equally to this work.

Key words: inectious diarrhea, ion absorption, enteric pathogens, mechanisms o diarrhea, EPEC

Daha causd by ntc nfctons s a majo facto n

mobdty and motalty oldd. An stmatd 24 bl-lon psods of nfctous daha occu ach ya and a

spcally palnt n nfants. Ths hghlghts th cl-

lula and molcula mchansms undlyng daha assocatdth th th classs of nfctous agnts, .., bacta, uss

and paasts. Sal bactal pathogns ha bn chosn as

modl ogansms, ncludng Vibrio cholerae as a classcal xamplof sctoy daha, Clostridium difcile and Shglla spcs as

agents of inammatory diarrhea and selected strains of patho-gnc Escherichia coli(E. coli) to dscuss th cnt adancs n

altaton of pthlal on absopton. Many of th cnt stud-s addssng pthlal on tanspot and ba functon ha

bn cad out usng uss and paasts. H, focus on

the rapidly developing eld of viral diarrhea including rotavirus,

noous and astous nfctons. Fnally dscuss Giardia

lamblia and Entamoeba histolytica as xampls of paastc da-ha. Paasts ha a gat complxty than th oth patho-

gns and a capabl of catng molculs smla to thos po-ducd by th host, such as sotonn and PGe

2. Th undlyng

mchansms of nfctous daha dscussd nclud altatons

n on tanspot and tght junctons as ll as th ulnc fac-tos, hch alt ths pocsss th though dct ffcts

or indirectly through inammation and neurotransmitters.


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or delivery o the A subunit into the cell.3 The A subunit ADP-ribosylates a GTPase, which regulates adenylate cyclase resultingin elevated cAMP production4 (Fig. 2). The production o cAMPactivates PKA, which then phosphorylates the regulatory domaino CFTR.5 While CT is the most dangerous part o V. cholo-raesarsenal, most o the modern work with CT actually providesinsights into understanding key cellular mechanisms. For exam-ple, by studying CTs retrograde translocation and the eventual

release o the A1 peptide into the host cytoplasm, the detailso endogenous retrograde trafcking have been uncovered.6 Inaddition, movement o CT through the Endoplasmic Reticulum-Associated protein Degradation (ER AD) pathway has shed lighton the way cells deal with misolded proteins in the ER.7 Asiderom the knowledge acquired in these studies, the CT B-subunithas shown great promise as an adjuvant in a number o recentvaccine studies. Although there have been additional insightsinto the transporters aected by CT. In addition to increased Cl-secretion, the absorption o Na+ is decreased through a cAMP-dependent mechanism where the activity o both apical sodiumtransporters, NHE2 and NHE3, is decreased8 (Fig. 2). Together,this leads to an increase in NaCl levels in the intestinal lumen by

enhancing secretion or decreasing absorption.In addition to CT, V. choleraeencodes several other toxins,

which modulate ion secretion and perturb barrier unctionto cause massive diarrhea. The toxins that aect ion secretiondirectly include accessory cholera toxin (ACE), which stimulatesCa2+ dependent Cl- secretion; NAG-stable toxin, which activatesguanylyl cyclase, thus stimulating cGMP production, whichleads to PKG-mediated activation o CFTR; and, fnallyV. chol-erae cytolysin (VCC), which creates anion permeable pores9-11(Fig.2). One o the phenotypes associated with VCC toxicity hasbeen linked with the newly described phenomenon autophagy.12

the pathophysiology o inectious diarrhea and explore the poten-tial o novel anti-diarrheal drugs.

Pathophysiological Mechanisms of Infectious

Diarrhea

Bacterial diarrhea. In developing countries, enteric bacterialpathogens and parasites are the leading cause o inectious diar-

rhea. Although even in the United States, the requency o bac-teria-induced illnesses is considerably high. EnterohemmorhagicE. coli(EHEC) inection causes disease in approximately 75,000people per year,1 whereas C. dicileremains the major cause ohospital acquired inections.2 Bacterial diarrhea ranges in dura-tion rom a ew hours or some released toxins to several weeksor active inections o enteroaggregative E. coli. Here we use V.choleraeas an example o secretory diarrhea and then discuss C.dicile-associated diarrhea, which relies on neuropeptides andinammatory mediators or pathogenesis. In addition, Shigellaspecies are used as an example o inammatory and invasive diar-rhea while E. coli have a variety o strategies, one o which isthe reduction in absorption both through a loss o microvilli and

through a direct eect on ion transporters. These our groups opathogens have been selected to explain the major mechanismsinvolved in diarrhea.

Vibrio cholerae. Despite being the ocus o scientifc studysince the 1800s, V. choleraeremains a threat today. While peoplewith access to treated water are typically not exposed, areas with-out adequate chlorination or fltration still suer rom epidemiccholera. V. choleraehave several toxins, which are used to com-promise the host, with the most important o these being choleratoxin (CT) itsel. CT consists o an A subunit bound to a pen-tameric ring o B subunits, where the B subunits are responsible

Figure 1. An o of gnal mchansms causng daha. At th most basc ll, daha s causd by ncasd scton o dcasd

absorption of uids and electrolytes. Certain ion transport processes are particularly associated with diarrhea. These include CFTR and CLCA, which

a chlod channls and th Na+/H+ xchang sofom, NHe3, hch s nold n Na+ absopton. Altatons n tght junctons cat an mpotant

pathay fo th momnt of both ons and at. DrA s sponsbl fo chlod absopton and s assocatd th congntal chlod daha.Data on aquapons s lmtd but thy a xpctd to contbut to daha hn absopton s ducd. SGLT-1 tanspots sodum and glucos and

s tghtly coupld th at momnt and s th bass fo oal hydaton usng glucos to nhanc sodum absopton.


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in the presence o the actin cross-linking domain o RTX. Actinand myosin light chain play a critical role in the regulation otight junctions by orming a belt-like structure around the cell,

which can be tightened to increase paracellular ow. In this case,the unctionality o the junctional actinomyosin ring is lost dueto the sequestration o actin in incorrectly polymerized aggre-gates. The third toxin, Zot, which also impacts barrier unction,serves as both a phage assembly protein and an enterotoxin.16This is similar to what is seen with Ace, which is also a phagestructural protein.17 Many o the virulence actors associated withV. choleraeare part o an integrated prophage called , which iscomposed, in part, o Ace and Zot.18 Zot is not unctional in itsphage associated orm but is instead cleaved into an active 12kDa peptide.16 The Zot peptide binds to an apical receptor or ahost protein called zonulin, which regulates permeability exclu-sively in the small intestine.19 Loss o barrier unction occurs only

in rabbit ileum but not in colon, consistent with the localizationo the zonulin receptor. Recently, a smaller ragment o Zot con-sisting o only 6 amino acids was shown to be capable o causingthe same change in resistance as well as causing the dissociationo ZO-1 rom tight junctions.20 However, it should be noted thatthis work is considered controversial by some. Thus, V. choleraehas three dierent toxins that promote secretion and three toxinsthat promote the loss o barrier unction and, in combination,this results in a particularly severe diarrhea (Fig. 2).

C. difcile. C. dicile is the leading cause o nosocomialdiarrhea and accounts or almost all cases o pseudomembranous

The VCC toxin causes large vacuoles to orm in host cells inaddition to its cytolytic eect on red blood cells. While themechanism behind this vacuole ormation is only beginning to

be understood, recent studies have shown that VCC is associatedwith double membrane vesicles and LC3-II accumulation whichare characteristic o autophagy. In addition, mouse embryonicfbroblasts defcient or Atg-5 (which is needed or LC3 conver-sion) or inhibitors o autophagy including 3-methyladenine blockthe ormation o vacuoles in response to VCC. While vacuoleormation appears to be a severe phenotype, it is actually a pro-tective cellular response and its inhibition results in a more than50% decrease in cell survival ater toxin exposure. In this case,vacuole ormation appears to be a part o the natural removal oVCC rom the cellular membrane, blocking urther dysregulatedCl- transport and protecting the cell.

The V. choleraetoxins which alter intestinal barrier unction

include hemagglutinin/protease or HA/P, RTX and Zot. HA/P isan extracellular protease, which cleaves a tight junction structuralprotein, occludin, that is known to regulate paracellular perme-ability (Fig. 2).13 This results in the subsequent loss o the sca-olding protein ZO-1 rom the tight junction. RTX cross linksactin and induces cell rounding, loss o transepithelial resistance(TER) and an increase in permeability to FITC-dextran 3000.14Analysis o the crysta l structure o RTX cross-linked actin andmass spectrometry showed that residues E270 and K50 orm acovalent iso-peptide bond.15 Interestingly, the ungal toxin phal-loidin was able to partially restore the ability o actin to polymerize

Figure 2. Mchansms undlyng V. cholerae-induced diarrhea. Cholera is characterized by severe watery diarrhea due to changes in ion secretion

and absorption. Both CLCA and CFTR-dependent Cl- secretion are activated, the rst by Ace and the second by cholera toxin and NAG heat-stabile

toxn. incasd cAMP lls also block sodum absopton though NHe2 and NHe3. V. cholerae also cats anon pmabl pos though nston

of VCC. In concert with changes in ion transport, paracellular permeability is also decreased. Zot interaction with zonulin causes ZO-1 to dissociate

fom tght junctons hl HA/P clas occludn and rTX ntfs th th contactl actn ng.


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Earlier studies showed that TcdA causes direct alterations inbarrier unction and ion transport. For example, purifed toxinA has also been shown to cause net luminal accumulation osodium, chloride and potassium in rabbit intestine in vivo.30Ussing chamber studies using isolated mucosal strips rom guineapig ileum demonstrated that TcdA increases transepithelial per-meability and decreases electrogenic Na+ absorption while elicit-ing a Cl- secretory response.31 Rounding up o cells and barrierunction disruption corresponding with structural alterations operijunctional actinomysin ring occurred in human intestinalepithelial T84 cells exposed to TcdA.30,32

Besides the direct eects o the toxins, other mechanismsunderlyingC. dicileassociated diarrhea include inammation

and activation o neuropeptides. The C. diciletoxins initiate anextensive inammatory cascade that causes increased damage tohost tissues resulting in uid exudation. TcdA causes release oseveral proinammatory cytokines such as leukotriene, PGE

2, and

tumor necrosis actor (TNF in vivo).33 It also directly activatesmonocytes to release IL-1 and IL-6,34 and increase neutrophilmigration in vitro.35 Other toxin-mediated inammatory eectsinclude release o reactive oxygen species, activation o mitogen-activated protein kinases and NFB activation.33 A number ostudies suggest that important cellular responses to C. diciletoxins such as p38 MAP kinase activation, mitochondrial

colitis, which is an acute colitis characterized by ormation o anadherent inammatory membrane overlying the site o injury21(Fig. 3). The recent emergence o novel epidemic strains hascaused increased concern in clinical settings because antimicro-bial therapy predisposes patients to C. dicileassociated diar-rhea (CDAD).22 The pathogenic process oC. dicileinectionstarts with initial colonization ollowed by the production o twodistinct exotoxins, Toxin A and B (TcdA and TcdB), as well asan additional toxin called binary toxin (CDT) which is ound insome hypervirulent strains oC. dicile.23 TcdA binds eectivelyto the apical side o the host cell to glycoprotein gp96, whichorms part o the receptor in humans.24 In contrast, TcdB gainsaccess to the basolateral side o the cell ater tight junction dis-

ruption and binds preerentially to an unidentifed receptor25,26

(Fig. 3). TcdA and TcdB are potent cytotoxic enzymes that spe-cifcally glucosylate the small GTPase protein Rho, which leads todisruption o cytoskeletal integrity and cytotoxic eects.27 CDT,an actin-specifc ADP ribosyltranserase, potentiates the toxicityo TcdA and B and may increase the severity o CDAD. 28 Whileearly studies in animals implicate TcdA as the primary actor inthe pathogenesis oC. dicileinection, recent studies showedthat disruption o the tcdBgene led to a signifcantly attenuatedvirulence phenotype in the hamster model suggesting that TcdBmight play a key role in disease pathogenesis.29

Figure 3. Pathognss ofC. diffcile-assocatd daha. C. difcile poducs toxn A and toxn B (TcdA and TcdB). TcdA bnds to th apcal sd of

the cell and, af ter internalization, causes cytoskeletal modication and disruption of tight junctions. The resulting loss of epithelial barrier function

facltats TcdA and TcdB to coss th pthlum th pfntal bndng of TcdB to th basolatal cll mmban. Both toxns a cytotoxc and

lead to production of proinammatory cytokines, increase in vascular permeability, recruitment of neutrophils and monocytes, epithelial cell apoptosis

and connct tssu dgadaton, sultng n psudommban fomaton and daha. Futh, th actaton and las of aous nuopptds

by the toxins stimulates ENS to elicit uid secretion, causing diarrhea.


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are ound very rarely in developed countries and have a generallylow inection rate over all, however, S. dysenteriaecauses the mostlie-threatening o all o these inections due to the production

o Shiga toxin, which can lead to hemolytic uremic syndrome(HUS). While all our species o Shigella are invasive due to alarge virulence plasmid, there is some variation in the plasmidand S. fexneri is the best studied in this regard. The invasionprocess is complicated and occurs through a trigger mechanismon the basolateral side o epithelial cells ater the bacteria havealready passed through M-cells and potentially, macrophages.41In addition, Shigella have actin tails and are capable o cell tocell spread, increasing their ability to colonize the epithelium.42The type III secreted eector proteins involved in this process arenumerous and have been recently and thoroughly reviewed bySchroeder and Hilbi.43

Invasion and infammatory response. Shigella causes an inam-

matory diarrhea and the cellular response to various steps o theinvasion process are the primary cause o inammation (Fig. 4).The destruction o macrophages ater emergence rom M-cellscauses an initial release o IL-1, which attracts PMNs.44,45PMNs release a precursor to the secretagogue adenosine, whichactivates Cl- secretion. This early step in inammation is exac-erbated by the presence o ree bacteria on the basolateral sideo cells, which allows access to toll-like receptors. Shigella LPSis capable o activating TLR4, although recent studies suggestthat it is about 50% less active than LPS rom E. coli in acti-vating NFB due to a reduced level o acetylation.46 Thereore,

damage and IL-8 release occur prior to and independently o Rhoglucosylation.33

Another striking eature oC. dicletoxin-associated intes-

tinal responses is activation o enteric nerves and enhanced pro-duction or release o neuropeptides including Substance P (SP),calcitonin gene-related peptide (CGRP) and neurotensin,36-38which are known to elicit Cl- secretion in intestinal epithelialcells. The antagonists o the Substance P receptor, Neurokinin-1(NK1) and calcitonin gene-related peptide (CGRP), block uidaccumulation and mannitol ux in response to toxin A.33 Alsoin a NK1-R-/- mouse ileal loop model, TcdA-mediated luminaluid accumulation was considerably reduced.39 Thereore, unlikecholera toxin and E. colienterotoxins, which trigger intestinalsecretion without intestinal inammation, the pathophysiologyoC. dicile-associated diarrhea involves a necroinammatoryreaction, which activates mast cells, nerves, vascular endothe-

lium, and immune cells in addition to impairment o tight junc-tions (Fig. 3). Further studies pertaining to detailed analysis othe contribution o the electrogenic versus electroneutral com-ponents o ion absorption in the well-established animal mod-els oC. dicileinection would beneft the modeling o diseasepathogenesis and help elucidate the mechanisms o C. dicileassociated diarrhea.

Shigella species. There are our major Shigella species thatcause diarrheal disease. The most common species in the U.S.and other developed countries is S. sonnei ollowed by S. fex-neri.40 Two other Shigella species, S. dysenteriae and S. boydii,

Figure 4. Invasion and inammation caused by Shigella. Shigella species cross the epithelial barrier through M-cells where they encounter and elimi-

nat macophags. Bndng of lpopotn to TLr2 sults n th poducton of th chmoattactant iL-1. Aft tanslocaton though M-clls LPS can

bnd to basolatal TLr4 hch causs th poducton of iL-6 and iL-8. Ths ffct s somhat dmnshd du to th actylaton of LPS n Shglla. iL-8

s a potnt chmoattactant fo PMNs and s also poducd du to actaton of ntacllula Nod1 by pptdoglycan. PMNs a th pmay dstuct

force in Shigella infection. PMNs cause Cl - scton though gnaton of a pcuso to th sctagogu adnosn and can also caus ulcaton of

the epithelium, which results in a decrease in the absorptive surface but also maximizes permeability and allows easy access of gut ora to the basolat-

eral surface of cells further driving inammation.


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periplasm to the bacterial cytoplasm.48 Miceinected with an MppA mutant were able toclear an otherwise lethal dose oS. fexneri.48Masking the molecular patterns is not anentirely eective strategy, thereore, Shigellahave additional strategies that block signaltransduction ater pathogen recognition.

Type III secreted eector proteins modulateinfammation. Shigella have a type III secre-tion system, which injects a number o eectorproteins required or initial invasion, escaperom the vacuole and movement within thecell. Because shigellosis is primarily due toinammation, bacterial proteins that modu-late the host immune response can dieren-tially modulate the host diarrheal responseas well. There are three eector proteins thatmeet this criteria: OspF, OspG and IpaH(Fig. 5). The frst, OspF, has a unique wayo inhibiting MAP kinase signaling. It is an

entirely new type o enzyme which does notexist in mammalian cells called a phospho-threonine lyase.49 OspF is capable o dephos-phorylating threonine residues in a way thatnot only removes the phosphate group butalso the oxygen atom, which is normally parto the amino acid, as well as a hydrogen atomrom the adjacent carbon. In doing so it cre-ates a carbon-carbon double bond within thebackbone o the amino acid. In this ormthere is no hydroxyl group capable o beingphosphorylated, thus the process is essentially

irreversible. Several MAP kinases are aectedby OspF, including Erk1/2, p38 and JNK.49These kinases activate transcription actorswhich control the production o proinam-matory cytokines including IL-1, IL-6 and

TNF. MAP kinases are also targeted by IpaH which is a ubiq-uitin ligase.50 This was discovered using a yeast model wherethe yeast target was Ste7, a MAPKK. Ste7 is actively targetedor proteasomal degradation ater being ubiquitinated by IpaH,thus impairing urther MAP kinase activation and transcrip-tion o proinammatory cytokines. In contrast, OspG activelyintereres with the ubiquitination o phospho-IB, prevent-ing its degradation.51 IB normally binds to NFB and pre-

vents its translocation to the nucleus. Under normal conditionsIB phosphorylation leads to its ubiquitination and subsequentdegradation allowing NFB translocation and transcriptionalactivation. OspG is autophosphorylated, leading to the associa-tion with ubiquitin conjugating enzymes such as UbcH5 andUbcH7.51 While it is clear that OspG eectively blocks phospho-IB ubiquitination and degradation, it is unclear whether IBis a specifc target. The ability o OspG to interact with multipleubiquitin-conjugating enzymes suggests that there may be addi-tional targets, but the presence o IpaH suggests that at least someubiquitin-mediated degradation is let intact. OspF, IpaH and

to some extent, Shigella actively evade TLR4 recognition, whileTLR2 is activated by Shigella through lipoproteins.47 In addition,the interaction o Nod1 with shed peptidoglycan rom intracel-lular bacteria also leads to NFB activation and IL-8 produc-tion.48 IL-8 is another pro-inammatory cytokine, which attractsPMNs. In this case, PMNs cause many o the symptoms o thedisease but also lead to its eventual clearance.

Shigella are very closely related to E. coli and yet the host

response is considerably dierent due to their ability to cross theepithelium and access basolateral toll-like receptors in addition totheir ability to invade cells, where there are Nod proteins. Becauseo this, Shigella species have evolved a number o proteins, whichcounteract the host immune response. One method is preventa-tive with mechanisms such as the alteration in LPS acetylation,which reduces TLR4 activation.46 In addition, Shigella along withseveral other types o bacteria have been shown to have a recyclingmechanism or their peptidoglycan, which is designed to preventits release and interaction with Nod1. There are two permeases,AmpG and MppA, which transport ree peptidoglycan rom the

Figure 5. Modulation of pro-inammatory signaling by Shigella T3SS effectors. Several type

iii sctd ffcto potns ha bn shon to modulat th host mmun spons dung

Shigella infection. The rst is OspG which interferes with the ubiquitination of phosphory-

latd iB, pntng ts dgadaton, thby ffctly blockng NFB tanslocaton nto

th nuclus. in contast, ipaH actually nducs ubqutnaton and dgadaton of MapKKs,preventing further cascade activation. Finally, OspF has a unique way of cleaving the phos -

phat goup and a f xta atoms fom MapKs ncludng ek1/2, JNK and p38 hch pnts

futh phosphoylaton.


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o pathogenic E. colistrains responsible or diarrheal outbreakshave been recognized. These include enteropathogenic E. coli(EPEC), enterohemorrhagic E. coli(EHEC), enterotoxigenic E.coli(ETEC), enetroaggregative E. coli(EAEC), enteroinvasive E.coliand diusely adherent E. coli(DAEC). This article ocuseson diarrheagenic E. colistrains aicting humans, in particularEPEC, which has been studied extensively in recent years, to gain

understanding o the mechanisms underlying the pathophysiol-ogy o early diarrhea. A brie introduction regarding ETECinection is provided primarily or comparison with these moremodern studies.

Enterotoxigenic E. coli. ETEC causes toxigenic secretorydiarrhea characterized by massive intestinal uid secretion. Thekey virulence attributes o ETEC include adherence to epithelialcell suraces by colonization actors and elaboration o heat labile(LT) and heat stable (ST

a) enterotoxins. Some strains o ETEC

may also express Entero-aggregative heat stable toxin 1 (EAST1).Similar to cholera toxin, heat labile toxins elicit increases in Cl -secretion via activation o cAMP. ST

a, however, is known to evoke

secretion and diarrhea by elevation o intracellular cGMP. Ater

binding to its receptor, guanylyl cyclase C (GC-C), STa throughcGMP dependent pathways is known to stimulate CFTR translo-cation to the surace o villus enterocytes causing its activation.57There is net Cl-, HCO

3-, and water secretion as well as inhibition

o Na+/H+ exchange in jejunal enterocytes by STa.58 Other studies

suggested that STa

is only anti-absorptive and does not stimulateCl- secretion.59 Although GC-C has been shown to be the pri-mary receptor involved in ST

amediated secretory response, recent

studies have shown that a non-GC-C receptor exists in the mouseproximal intestine and unctionally stimulates ST

a-induced duo-

denal bicarbonate secretion through a mechanism independento CFTR.60 The CFTR-independent mechanism is urther sup-

ported by studies demonstrating stimulation in HCO3-

secretionin the duodenum o CF patients61 and CFTR knock-out mice.62Whether this mechanism is relevant in normal individuals oroccurs as an adaptive response due to the loss o CFTR needsto be investigated.60 Further elucidation o alternative bicarbon-ate secretory mechanisms modulated by ST

autilizing DRA and

PAT-1 knock-out mice would a lso be o interest.LT has two serogroups, LTI and LTII, that do not cross react

immunologically. LTI is expressed by strains o E. colithat arepathogenic or both animals and humans, whereas LTII is rarelyound in human isolates and has not been associated with dis-ease. Both LTI and LTII closely resemble CT structurally andunctionally. LTI is composed o an enzymatic A subunit and a

pentameric B subunit that binds strongly to ganglioside GM1.Ater binding to the membrane, LTI is endocytosed and under-goes trafcking through the trans-Golgi network (TGN).63 LTtargets adenylate cyclase leading to increased cAMP, activationo PKA leading to increased phosphorylation and activation oCFTR. The resulting stimulation in Cl- secretion is a classicalexample o how LT and CT cause diarrhea. LT has been shownto decrease H+/PEPT co-transporter through a cAMP dependentpathway in Caco-2 cells.64 Thus, cAMP mediates the hypersecre-tory eects o LT resulting in decreased absorption and increasedsecretion o uids and electrolytes.

OspG interere with both the MAP kinase cascade and NFBtranslocation, blocking many o the critical pathways required ortranscriptional activation o host cytokines.

PMN activation can also lead to a more generalized loss oabsorptive unction through the destruction o the epitheliallayer. In the case o Shigella, this actually enhances bacterialinvasion by allowing greater access to the basolateral surace o

cells.52 However, it has been recently shown that Shigella activelyopposes this loss o the epithelium through the interaction oOspE with the host protein integrin-linked kinase (ILK).53 Thisinteraction slows down turn over o ocal adhesions, although themechanism is not due to increased kinase activity but, instead,increased membrane association o ILK. While this mechanismprevents the initial loss o cells, it also prevents migration inwound healing assays so tissue which is already damaged will notheal. Studies o rectally inected guinea pigs suggest that OspEhelps promote greater colonization, more severe diarrhea, inam-mation and hemorrhaging. Maintaining epithelial integrity inthis manner is somewhat unusual considering the disruption ocellular architecture caused by other pathogens; however, because

Shigella colonize the epithelium intracellularly loss o cells whichare already colonized could be detrimental to the bacteria.

Alterations in ion transport and barrier unction. While diar-rhea caused by Shigella is primarily due to host inammatoryprocesses and many Shigella eector proteins actively aectinammation, there are additional actors that directly alter iontransport and tight junctions. Viable S. fexneri, in contrast tobacterial supernatant, heat killed bacteria or purifed LPS is capa-ble o decreasing transepithelial resistance.54 A number o tightjunction proteins including claudin-1, ZO-1, ZO-2 and occludinare altered by the presence o Shigella; however a specifc eec-tor protein responsible or these changes has yet to be identifed.

Shigella also possess three enterotoxins known as SigA, SepAand Pic, which are serine protease autotransporters or SPATES.As the name suggests, the primary unction o SPATES is pro-teolytic. The autotransporter portion o the name reers to theexit mechanism rom the bacterial outer membrane through a-barrel pore ormed by the C-terminus o the protein itsel.While Pic has been associated with the cleavage o mucin and isbelieved to play a role in colonization, only SigA plays a clear rolein uid accumulation.55 These proteases are also associated withvarious cytotoxic eects and induce cell rounding. The actualmechanism involved in SigA-mediated uid increases in rabbitilleal loops has not been determined. A homologous protein inenteroaggregative E. colicalled Pet has been shown to degrade

spectrin, a cytoskeletal component, and cause changes in shortcircuit current in addition to cell liting. So, while Shigella doesproduce toxins which alter uid movement, and proteins whichalter tight junction regulation, they are considered less importantthan inammation in causing diarrhea.

Escherichia coli. E. coli is the most abundant acultativeanaerobe o the human colonic ora and typically colonizes thegastrointestinal tract within ew hours ater birth.56 Usually, com-mensal E. coliinteract with the host in a mutually benefcial way;however, some strains oE. coliacquire virulence attributes thatcan cause a broad spectrum o disease. Several dierent classes


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that while absorption o Na+ ions is important, it is not directlycoupled with water movement in the case o NHE2. Studies uti-lizing EPEC mutants revealed that NHE3 inhibition is depen-dent on an intact T3SS III and occurs via the eector moleculeEspF.75 EPEC-induced inhibition o NHE3 activity also requiresNHE regulatory actors (NHERF) that are important or regula-tion o NHE3 unction and surace expression.76 The inhibitory

eect o EPEC on NHE3 is enhanced when PS120 cells are co-transected with the scaolding/regulatory proteins NHERF1and NHERF2.75 In addition to NHE3, EPEC has been shownto rapidly inactivate sodium-D-glucose transporter (SGLT1), themajor contributor o uid uptake in the small intestine.77 Dueto the critical role o SGLT1 in oral rehydration therapy, urtherresearch into the mechanism o this process would be o greatbeneft (Fig. 6).

Cl--HCO3

-(OH-) exchangers unction in concert with theNa+/H+ exchangers in mediating coupled electroneutral NaClabsorption in ilea l and colonic epithelial cells. Parallel to EPECmediated inhibition o NHE3, a decrease in apical Cl -/OH-exchange activity was demonstrated in Caco-2 and T84 cells.68

Further studies to delineate the mechanisms underlying thedecrease in Cl-/OH- exchange revealed an important role or theT3SS. Mutations in either the eector protein espGor its para-log espG2partially attenuated the inhibitory eects o EPEC,whereas the double mutant completely abolished the inhibitiono Cl-/OH- exchange activity.68 Since EspG and EspG2 play animportant role in disrupting the host microtubule network,78these studies suggested that EPEC-induced inhibition o Cl-/HCO

3-(OH-) exchange activity is dependent on the disruption

o microtubules. This was urther confrmed by the observa-tion that colchicine and nocodazole, microtubule-disruptingagents, decreased Cl-/OH- exchange activity in Caco-2 cells.68

Among the candidate genes or luminal human intestina l Cl

-

/HCO3

- exchangers are two members o the SLC26 gene am-ily: SLC26A3, or DRA, and SLC26A6, or PAT-1 (putativeanion transporter-1). DRA is predominantly expressed in thecolon and plays a major role in the apical Cl -/HCO

3- exchange

process based upon its implication in congenital chloride diar-rhea (CLD).79 CLD is a rare genetic disorder characterized byvoluminous diarrhea, massive loss o Cl- in stool and metabolicalkalosis. In addition, in contrast to PAT-1 knock-out mice,DRA knock-out mice develop a robust diarrheal phenotype aswell as serum electrolyte imbalances.80,81 Both biochemical andimmunouorescence studies to assess surace expression o api-cal anion exchangers demonstrated reduced levels o DRA (but

not PAT-1) on the apical plasma membrane in response to EPECinection. This reduction in plasma membrane levels o DRAwas EspG/EspG2 dependent.68 An important role o DRA inthe pathophysiology o diarrhea was urther confrmed in vivo.A marked redistribution o DRA rom apica l to subapical com-partments was demonstrated in EPEC-inected mouse colon.68Furthermore, mice challenged with C. rodentium, the mouseequivalent o EPEC, showed 10 old downregulation o DRAand atal uid loss.82,83 A decrease in DRA and NHE3 activitiesimpairs luminal NaCl absorption and underlies the pathophysi-ology o EPEC induced early diarrhea (Fig. 6).

Enteropathogenic E. coli(EPEC). EPEC is a major cause opersistent, watery diarrhea in inants, oten accompanied by low-grade ever and vomiting. The pathogenic mechanisms o EPECremained elusive or many years because, in contrast to prototypicenteric bacterial pathogens, EPEC does not produce classicalenterotoxins such as LT in order to inuence host cell pathways.In addition, EPEC is typically regarded as non-invasive in com-

parison to bacteria such as Shigella and Salmonella, although alimited number o the bacteria are internalized. However, EPECdoes encode a T3SS and produces a characteristic attaching andeacing (A/E) lesion which is marked by eacement o microvillion the epithelial surace at the site o bacterial attachment.65 Inaddition, there is an accumulation o cytoskeletal proteins beneathadherent microcolonies leading to actin cup or pedestal orma-tion, depending on cell type. This proound change in intestinalepithelial cel ls induced by A/E lesions contributes to the diarrhealphenotype due to loss o overall absorptive surace.66 However,diarrhea occurs as quickly as 34 h ater the ingestion o thepathogen, suggesting that mechanisms other than malabsorptionare at work.66,67 Progress is being made in unraveling the basis o

EPEC pathogenesis at the molecular level and the mechanism(s)by which EPEC alters host epithelial responses are beginning tounold. Studies over the past decade suggest that EPEC-induceddiarrhea is a multi-actorial process and involves several actorsincluding disrupted paracellular permeability and disturbancesin ion transport. Recent advances suggest that EPEC has a directeect on ion transport processes notably those related to solute,electrolyte, serotonergic and short chain atty acid transporters.The ollowing section will review potential mechanisms underly-ing EPEC-associated diarrhea.

Alterations in Na+ and Cl- transport. Investigation o the directeects o EPEC on intestinal ion transport suggest that decreased

intestinal NaCl absorption underlies the pathophysiology o theearly onset o diarrhea rather than an increase in Cl- secretion.68-70Earlier studies utilizing human intestinal epithelial Caco-2 cellsshowed that EPEC inection resulted in a small and transientincrease in Cl--dependent short circuit current (I

sc), a measure

o charged ion movement.71 In contrast, in T84 cells, a coloniccrypt cell line widely used to study apical Cl- secretion, there wasno increase in Cl- secretion but rather an inhibition o agonist-induced secretory responses.70 Although secretion is minimallyaected, EPEC alter Na+ and Cl- absorption in both Caco-2 andT-84 cells.69 While net Na+ uptake mediated by Na+/H+ exchang-ers (NHE) is actually increased by EPEC in Caco-2 cells, theactivity o NHE isoorms is dierentially modulated.69 In the

intestine, both NHE2 and NHE3 isoorms are apically local-ized, while expression o NHE1 is restricted to the basolateralmembranes. Notably, EPEC inection o intestinal epithelial cellsdecreases the activity o NHE3, the major Na+-absorbing iso-orm in the small intestine. In contrast, NHE1 and NHE2 iso-orms are stimulated in response to inection with EPEC, whichis thought to be a compensatory host response to alteration inNHE3 activity.69 However, NHE2 knock-out mice do not havethe same intestinal absorption deects or a diarrheal phenotype asNHE3 knock-out mouse mice.72,73 Additionally, NHE2 is unableto prevent diarrhea in NHE3 knock-out mice.74 This suggests


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secretory mechanism causing diarrhea.86 Recent studies demon-strated that EPEC inection o Caco-2 cells decreases SERT unc-tion.87 Protein tyrosine phosphatases (PTPases) were involved ininhibiting SERT in Caco-2 cells inected with EPEC. DecreasedSERT expression and mucosal 5-HT content also occurred inEPEC-inected murine small intestine.87 Future identifcation othe EPEC eector molecule(s) that activates PTPases and medi-ates inhibitory eects on SERT will be o importance in develop-ing new targets to modulate the serotonergic system in treatmento inectious diarrheal diseases.

Another potential conributor to EPEC-induced diarrhea is themodulation o butyrate absorption mediated by monocarboxylate

transporter (MCT1) in the intestine.88

Butyrate, the major shortchain atty acid, provides energy or colonocytes and is knownto stimulate electrolyte absorption.88 Butyrate transport in epi-thelial cells is reduced by EPEC inection via a TTSS dependentmechanism and occurs via decreasing the levels o MCT1 onplasma membrane88 (Fig. 6).

Disruption o tight junctions and immune responses to EPEC.Several lines o evidence demonstrate that EPEC inection ointestinal epithelial cells disrupts the tight junction barrier dueto activation o myosin light chain kinase (MLCK) and dephos-phorylation o occludin.89,90 These eects are dependant on a

Water transport. Aside rom modulating electrolyte transport,C. rodentium has been shown to directly impact water transport.DuringC. rodentium inection, water channels called aquaporins2 and 3 (AQP2 and AQP3) redistribute rom cell membranes tothe cytoplasm partly due to EspF and EspG.84 This altered AQPlocalization correlates with increased uid levels in internal stoolalthough the mice do not exhibit diarrhea per se. This loss owater absorption is thought to be a contributing actor to diar-rhea during EPEC inection. In addition, genome-wide transcrip-tional array studies utilizingC. rodentium inected mouse colonshowed a drastic downregulation o AQP8 mRNA83 (Fig. 6).

Modulation o serotonin and butyrate transporters. EPEC is also

known to alter other epithelial processes that indirectly inuenceelectrolyte transport, epithelial integrity and immune responsesin intestinal epithelial cells. 5-HT, a neurotransmitter and hor-mone o the GI tract, aects several physiological processesincluding absorption and secretion o uids and electrolytes viaserotonin receptors.85 5-HT is internalized through the highlyselective Na+ and Cl--coupled serotonin transporter, SERT, whichacilitates 5-HT degradation by intracellular 5-HT catabolizingenzymes. Thereore, SERT regulates 5-HT content and availabil-ity in the gut lumen. Enterotoxins such as CT are known to causethe release o serotonin in the small bowel which supports the

Figure 6. EPEC-induced early diarrhea is multifactorial. EPEC infection of human epithelial cells decreases epithelial ion absorption to cause diarrhea.

The type III secretion system of EPEC releases E. colisctd potns (esps) nto th host cytosol. espF nhbts th functon of Na+/H+ xchang

isoform 3, whereas EspG, via disruption of microtubules, decreases surface levels of DRA leading to a decrease in apical Cl -/OH -(HCO3-) xchang

activity. The resulting inhibition of coupled, electroneutral NaCl absorption in the infected intestine is thought to contribute to diarrhea. EPEC, via an

unknown effector molecule, decreases butyrate absorption by decreasing the plasma membrane expression of monocarboxylate transporter 1. EPEC-

nducd actaton of potn tyosn phosphatass (PTPass) dcass th functon of sotonn tanspot (SerT) and ncass 5-HT aalablty,

which can further affect ion absorption and motility to cause d iarrhea. Furthermore, SGLT-1 inhibition by EPEC is thought to promote uid accumula-

ton causng aly daha.


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contaminated bee, EHEC colonization o ruminants is actuallyasymptomatic because o dierences in Gb3 receptor distribu-tion. CD77/Gb3 is present on kidney glomerular cells in humansbut not in cattle, making HUS a serious complication only orhumans. Interestingly, human intestinal epithelial cells do noteven express GB3 receptors,94,96 however, a novel binding site orStx has been described at the base o small intestinal crypts o

leiberkuhn in paneth cells.97With respect to mechanisms o diarrhea, both Stx1 and Stx2

elicit luminal uid accumulation in the intestine.98 In this regard,purifed Stx1 inoculated into adult rabbit ileum induces uidaccumulation and is associated with apoptosis in villus absorp-tive cells.99 Similarly, Stx2 holotoxin evokes uid accumulationin vivo and inhibits net absorptive water transport across humancolon in vitro.98 Recent studies utilizing T84 intestinal cells and arabbit model o EHEC inection demonstrate a novel mechanismo Stx-mediated diarrhea. Stx1 inection decreases the secretiono galectin-3, a-galactoside-specifc lectin, resulting in mistar-geting o several important brush border proteins.100 Particularly,trafcking o vill in, NHE2 and dipeptidyl peptidase is impaired,

which is expected to a lter epithelial absorption, leading to diar-rhea.100 Interestingly, decreased galectin-3 levels have also beendescribed in Crohns disease.101 Whether EHEC virulence actorsencoded by T3SS directly aect epithelial ion transport processesand contribute to diarrheal phenotype o this pathogen, as in thecase o EPEC, requires urther investigation.

Viral diarrhea. Viruses including norovirus, sapovirus, ade-novirus, rotavirus and astroviruses are responsible or 3040%o acute episodes o diarrhea in the US.40,102 Compared to bacte-rial inections, many viral inections are mild and sel-limited.Norovirus inection aects people o all ages and accounts or40% o non-bacterial diarrheal outbreaks in the US.103 In con-

trast, rotavirus causes lie-threatening gastroenteritis in children,accounting or 35% o the hospitalized cases o diarrhea in theUS.104 In the ollowing section, we discuss three viral species thatcause diarrhea, including rotavirus, which has the only knownviral enterotoxin, as well as norovirus and astrovirus which areonly beginning to be understood.

Rotavirus. Rotavirus is the leading cause o lie-threateningdiarrheal diseases among young children. Research over the pastseveral years has provided important insights into mechanismo viral pathogenesis and led to successul development o live,attenuated vaccines or gastroenteritis.105 Rotavirus primarilyinects small intestinal villus cells and can cause watery diar-rhea without any signifcant intestinal inammation. The double

stranded RNA genome o rotavirus encodes or 6 structural pro-teins that orm virus particles (Vps) and 6 non-structural proteins(NSPs).106 NSP4 is the frst described virus-encoded enterotoxinand has been suggested to play a critical role in uid and elec-trolyte secretion.107 Interestingly, NSP4 is absent in the matureinective virion and is synthesized in inected villus enterocytes(Fig. 7). NSP4 and virus particles are released through the apicalmembrane o polarized epithelial cells by a non-classical secretorypathway.105 However, NSP4 is also released rom the basolateralside o inected enterocytes, although the role o basolaterally-released NSP4 in diarrhea is not clearly understood108 (Fig. 7).

unctional T3SS and several eector proteins including EspF,NleA and Map. The eect o NleA on tight junction integrityoccurs via inhibition o host cell protein trafcking throughCOPII-dependent pathways, while the mechanism o EspF activ-ity remains unknown.90 The decrease in TER seen with EPECinection also occurs in vivo in ileal and colonic mucosa.91 Thisparticular phenotype is EspF-dependent and correlates with the

redistribution o occludin, replicating in vitro data.91 Disruptiono tight junctions increases paracellular permeability allowingelectrochemical gradients to reach an equilibrium and has beenshown to have a synergistic eect with altered ion transport incausing diarrhea.

Initiation o the inammatory response is another eecto EPEC inection. EPEC is known to activate NFB, whichstimulates the transcription o IL-8, a prototypical cytokine thatrecruits PMNs to the site o inection.92 EPEC-induced activationo NFB also upregulates expression o the galanin-1 receptor,which results in increased Cl- secretion and uid accumula-tion within the lumen ater activation by its ligand, galanin-1.66Although inammation is certainly the net eect o EPEC

inection, the disease is not as inammatory as other bacterialpathogens such as Shigella and recent studies have demonstratedthat EPEC eector proteins dampen the inammatory responsesomewhat.93 The ability o EPEC to suppress the degree or sever-ity o the net inammatory response was suggested to be essentialor the survival o these noninvasive bacteria in the host.93

Collectively, these observations suggest that EPEC-induceddiarrhea is a multiactorial process with perturbation o electro-lyte, solute and water transport contributing to the developmento early onset diarrhea induced by EPEC inection (Fig. 6). Aprominent conclusion that emerged rom these studies is theuse o distinct eector proteins, suggesting selective eects o

EPEC on ion transporters. For example, EPEC inection o hostintestinal epithelial cells dierentially modulates NHE2 andNHE3 activities as well causes a decrease in plasma membranelevels o DRA but not PAT-1.68,69 Also, EspG which is involvedin disruption o the host microtubule network, is involved inperturbation o Cl- and water transport but does not modulateother apical membrane transporters such as Na+ or butyratetransporters.68,75,84,88

Enterohemorrhagic E. coli. EHEC is another major intestinalpathogen that is a subset o Shiga toxin producingE. coli(STEC).EHEC adheres to epithelial cells, expresses a T3SS and causes A/Elesions much like EPEC.63 Unlike EPEC, inection with EHECcan cause more severe symptoms including bloody diarrhea and

lie-threatening conditions such as hemolytic uremic syndrome(HUS) and thrombotic thrombocytopenic pupura. These symp-toms are largely attributable to the production o Shiga toxins(Stx1 and/or Stx2), both o which are composed o one 32 kDa Asubunit bound non-covalently to the pentameric ring o identicalB subunits. The A subunit possesses N-glycosidase activity thatcatalyzes depurination o a single adenine residue o 60S ribo-somes, rendering them inactive.94 The B subunits mediate thebinding o toxin to the globotriaosylceramide (Gb3) glycolipidreceptor present on the plasma membrane o certain eukary-otic cells.95 Although EHEC inections oten originate rom


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This was urther confrmed with studies demonstrating that netrotavirus-mediated uid transport was inhibited by treatmento mice with drugs that aect ENS unction.114 Further, clinicalstudies in hospitalized, rotavirus-inected children show that anenkephalinase inhibitor reduces diarrhea duration.115 IntracellularNSP4, however, is also known to increase intracellular calciumlevels through a PLC-independent mechanism.116 UtilizingNSP4-EGFP expression in HEK 293 cells, recent studies dem-onstrate that intracellular NSP4 causes actin reorganization in acalcium-dependent manner through decreased phosphorylationo the actin remodeling protein coflin.117 Modulation o subcor-tical actin dynamics and dysregulation o coflin inuences mem-brane trafcking events and ion transport processes.118

The Cl-

secretory component underlying the pathogenesiso rotavirus-associated diarrhea is complex, comprised o bothpro- and anti-secretory components.119 Unlike the purely secre-tory diarrhea caused by CT, rotavirus inection only moderatelyincreases luminal Cl- concentration.119 An increase in luminalCl- concentrations could be a consequence o decreased absorp-tion and/or increased secretion. Early studies demonstrated thatNSP4 can cause diarrhea in young mice, which is associatedwith Ca2+ mobilization and potentiation o cAMP-dependentuid secretion.107 Interestingly, in CFTR-defcient mouse pups,NSP4 continues to result in diarrhea ruling out the involvement

The virology and pathogenesis o rotavirus has been extensivelyreviewed recently.105

In contrast to classical secretory diarrhea, the viral entero-toxin, NSP4, induces diarrhea subsequent to maldigestion ocarbohydrates concomitant with decreased water absorption,increased Ca2+ mobilization and a relatively mild Cl- secretorycomponent (Fig. 7). Maldigestion o carbohydrates has been sug-gested as a major mechanism underlying the pathophysiology orotavirus-induced diarrhea. Rotavirus inection o Caco-2 cellsdecreases sucrose-isomaltase activity and apical expression in theabsence o enterocyte destruction, suggesting the involvement otrafcking mechanisms.109 Similarly, inection o young rabbitsor mice with rotavirus decreases disaccharidase activity.110,111 In

addition, NSP4 applied exogenously is known to induce Ca2+

release rom intracellular stores and plasmalemmal Ca2+ inuxthrough a phospholipase C-dependent mechanism.105 ThisNSP4-mediated Ca2+ mobilization can support diarrhea by inu-encing Ca2+-dependent epithelial processes such as ion transport,barrier unction or cytoskeletal regulation. Indeed, rotavirushas been demonstrated to increase paracellular permeability inCaco-2 cells.112 In addition, NSP4-mediated Ca2+ mobilizationmay trigger the release o amines/peptides as well as the release ocytokines, prostaglandins and reactive oxygen species, which canalone or collectively activate the enteric nervous system (ENS).113

Figure 7. Mchansms of otaus-mdatd daha. rotaus nfcton of ntocyts lads to us nty, fomaton of oplasms (vi) and

release of the virus and its toxin, NSP4. Intracellular NSP4 (iNSP4) induces an increase in intracellular Ca2+ pmaly though las fom er and a

PLC-independent mechanism. NSP4 released from the apical side increases intracellular calcium levels through a receptor-mediated PLC-dependent

mchansm. Th ncas n calcum by NSP4 dsupts mcollus cytosklton as ll as ba functon, ladng to an ncas n th paacllula

ow of water and electrolytes, causing diarrhea. The NSP4 induced increase in Ca2+ levels can also increase Cl - secretion directly through a CFTR-

ndpndnt mchansm and can caus las of amns, pptds, cytokns and act oxygn spcs, hch can stmulat th ntc nous

system indirectly to increase Cl - scton. Th basolatal las of NSP4 may also stmulat eNS. Maldgston of cabohydats du to a dcas

n sufac lls of sucas-somaltas and dcasd functon of SGLT-1 appas to b a majo mchansm undlyng daha causd by otaus

nfcton. NSP4 s xtacllula NSP4.


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One o these is the 3C-like proteinase (3CLpro), which issimilar to a previously described enzyme in rhinoviruscalled 3Cpro. 3Cpro intereres with eukaryotic translationby cleaving poly(A)-binding protein (PABP).121 Sinceboth ion transporters and tight junction proteins haveintermediate turnover times o 1218 hours on average,inhibition o host translation could limit the abundance

o these important regulators o intestinal homeostasis.Another mechanism which has the potential to intererewith host proteins involves the nonstructural proteinp48 o Norwalk virus. P48 binds to a cellular proteinknown as VAMP associated protein or VAP-A, whichassociates with v-SNARES, regulators o vesicle trafck-ing.122 As with the inhibition o translation, inhibitiono protein trafcking is likely to interere with cellularprocesses critical or maintaining absorptive unction aswell as barrier unction.

While cell culture models have been impossibleuntil recently and animal models are limited, a recentstudy was carried out using human intestinal biopsies

rom patients suering rom norovirus inection.123Initial observations showed that, in inected patients,villus length was decreased by 25%, while crypt lengthremained unchanged, reducing the overall absorptivesurace o the epithelium (Fig. 8). Miniature Ussingchambers (0.049 cm2) used to determine resistance, ux

and short circuit current on biopsies samples, showed an increasein I

scin patients inected with norovirus, which was dependent

on active Cl- secretion, consistent with CFTR activation. SGLT1activity was also increased suggesting that electrogenic Na+absorption is not impaired. In addition to changes in Cl- secre-tion, there was a marked decrease in transepithelial resistance

that corresponded with a decrease in the levels o occludin as wellas claudins 4 and 5. Because this work was carried out in humantissue, little is known about the mechanism which underlies thesechanges, however, recently developed norovirus cell culture mod-els should prove useul.

Astrovirus. Astroviruses, like noroviruses, are another causeo the stomach u. Astrovirus inection includes both diarrheaand vomiting and spreads in a similar manner to the previouslydescribed viruses. Little is known about the pathology o astro-virus inection as hospitalization is unlikely. One report is avail-able describing the pathology o an immune-suppressed 4 yearold male who showed villus shortening, which is consistent withwhat is seen in turkey models124 (Fig. 8). In addition, in both

these human biopsy specimens and in animal models, there isrelatively little inammation, suggesting that this is not animmune-mediated diarrhea.125 However, there is some evidencethat pro-inammatory pathways are activated in the cell. In act,activation o ERK1/2 is absolutely required or viral replication.126UV-inactivated astrovirus is also capable o activating ERK1/2with a similar response at 15 minutes post inection ollowed byinactivation at 30 minutes.126 The reduction in both viral proteinand RNA levels compared to controls is most consistent with thecase o HIV, which requires ERK activation or entry, however,the actual mechanism o ERK activation on adenovirus entry

o CFTR in uid accumulation.120 Unexpectedly, rotavirus inec-tion o rabbits actually stimulates Cl- absorption in intestinalbrush border membrane isolated rom villus cells and does notalter Cl- secretory responses in crypt cells.119 However, the netCl- secretory response is weak, suggesting that NSP4 exerts bothsecretory and anti-secretory actions to limit overall Cl- secre-

tion.

119

More in-depth studies are required to delineate the cel-lular mechanisms underlying rotavirus associated Cl- secretoryresponses. The potential role o apical Cl-/HCO

3- exchangers,

CLC chloride channels and key signaling events in the pathogen-esis o rotavirus inection would be o utmost interest.

Norovirus. Noroviruses, previously known as Norwalk-likeviruses, are a member o the Caliciviridae amily.40 They areone o a subset o viruses, which cause viral gastroenteritis, morecommonly known as the stomach u. This illness is typifed byshort term vomiting and diarrhea, both o which are inectious.The stomach u is caused by a large number o viruses rom sev-eral dierent amilies and includes sapoviruses, which are alsomembers o the Caliciviridae amily, a subtype o adenovirus, as

well as astrovirus. In contrast to rotavirus inection, none o theseviruses are particularly well-studied and inection rarely results inhospitalization, except in immune compromised individuals. Atypical norovirus inection lasts anywhere rom 13 days and notall inected individuals develop symptoms. Transmission is bothoodborne and person-to-person via inectious GI uids.

Research has been somewhat limited with regard to the molec-ular basis o norovirus induced diarrhea, since the virus has onlyrecently been grown in tissue culture and earlier studies relied ontransection o viral genes into host cells. Several viral actors caninterere with basic cellular unctions without causing cell death.

Figure 8. Shotnng of ll assocatd th al infctons. val nfctons

ncludng astous and noous caus llus bluntng o shotnng of th ll.

Th s a dcas n th numb of clls makng up th ll, ducng th oall

absopt sufac. intstnal pthlal clls a gnatd fom stm clls thn

th cypts and mo toad th llus tp, h thy a ultmatly shd. Blunt-

ng can b causd by ducd cll polfaton, although n th cas of noous

t s assocatd th an apcal llus nfcton, sultng n ncasd cll dath.

Villus blunting is also the cause of diarrhea associated with Celiac disease, which

s autommun ath than pathogn causd.


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Giardia is a non-invasive organism and trophozoites, the activeorm o the parasite, colonize the upper small intestine by adher-ing to the apical surace o the epithelium.130 Symptomatic inec-tion with Giardia causes acute or chronic diarrhea, dehydration,abdominal pain and malabsorption leading to malnutrition andweight loss. Also, this parasitic inection can trigger exacerba-tion o IBS131 and may cause development o post-inectious

unctional gastrointestinal disorders.132 Several studies utilizinga variety o cell systems, animal models and recent studies inhumans with chronic inections have provided important insightsinto the pathophysiology o giardiasis. An important fndingemanating rom these studies is that malabsorption, secretiono electrolytes and impairment o tight junctions may underliethe luminal uid accumulation during inection. Inections withGiardia species are known to cause a diuse shortening o themicrovillus brush border via activated T lymphocytes, accom-panied by the reorganization o F-actin and ZO-1 in enterocytesthrough MLC phosphorylation.133 Enterocyte apoptotic path-ways are also induced by G. lamblia via caspase-3 activation,which may also adversely aect epithelia l tight junctional integ-

rity.134 Previous studies rom both in vitro and in vivo animalmodels demonstrated decreased absorption o glucose and Na+and reduced disaccharidase activity due to the loss o epithelialabsorptive surace area.135,136 Also, G. lamblia-inected mouseintestine demonstrated secretion o Na+ and Cl- that was proteinkinase C dependent.137 However, the role o Giardia virulenceactors, such as proteinases and lectins, in modulating secretoryresponses needs to be ully determined.

The studies rom in vitro and animal models were recentlyconfrmed with observations rom human subjects inected withGiardia.138 It was established that G. lamblia inection is com-prised o active electrogenic anion secretion, impaired barrier dys-

unction and malabsorption. Analysis o tight junction proteinsin membrane extracts rom duodenal biopsy samples o inectedpatients showed a decrease in the epithelial tight junction proteinpool primarily caused by reduction in mucosal surace area.138However, ater mucosal surace area correction, only claudin 1expression was dierentially downregulated in chronic giardia-sis.138 Despite the decreased mucosal surace area, an increasein basal I

screecting increased Cl-/HCO

3(OH)- secretion was

observed in giardiasis.138 Importantly, the mucosal surace reduc-tion in human biopsy samples is predominantly loss o villussurace while crypts, an important site o active anion secretion,are not aected. Additionally, apoptosis and a dramatic reduc-tion in villus surace leading to impaired Na+-dependent glucose

absorption was observed in the duodenum o patients chronicallyinected with G. lamblia.133,138 Detailed investigation o the sig-naling mechanisms and virulence actors underlying epithelialdysunction ollowing inection will be o importance to under-stand the pathophysiology o giardiasis as well as development opost-inectious IBS.

Entamoeba histolytica.E. histolyticais a single cell protozoanwhich causes amebiasis. Pathologic eects are due to both para-site virulence actors and the host response to the parasite. In act,only about 10% o people inected with E. histolyticaactuallydevelop symptoms.40 In those who present with invasive disease,

and replication is not yet known. The activation o ERK in thesecells is moderate and more is known about the inactivation o theinnate immune response due complement inhibition. The astrovi-rus coat protein is capable o binding to the classical complementcomponent C1q, blocking the subsequent cascade o complementcleavage and membrane attack complex ormation.127

Astrovirus inection causes diarrhea with mild inammation

and has been shown to aect barrier unction. Moser et al.128demonstrated a decrease in TER in Caco-2 cells inected withastrovirus. Interestingly, there was also a loss o TER in cellswhich were treated with UV-inactivated astrovirus, which is inca-pable o replication, or with virus-like particles, which lack RNA.This suggests that the entry step is responsible or a loss o TERand is consistent with the act that other viruses are known totarget tight junctions during entry. However, the cellular recep-tor or astrovirus remains unknown. The authors also suggestthat there is a disruption o occludin in inected cells, however,cellular morphology is changed substantially ater viral inectionand is consistent with cells undergoing the early stages o apopto-sis. The alteration o occludin could represent an early apoptotic

event, although there is a loss in barrier unction which is notinuenced by early apoptotic events.

The association o astrovirus with cell death in humans is inquestion due to the lack o patient samples and the variabilityseen in animal models, specifcally turkeys and pigs. In vitro,however, cell death has been seen in Caco-2 cells starting at 36hours post-inection.129 This apoptosis is blocked by the caspaseinhibitor z-VAD-mk. Interestingly, this viral activation o thecaspase system is critical or the production o inectious virions.A precursor o the capsid protein called VP90 has a caspase cleav-age site, which is clipped as a step in generating an intermediatecapsid protein, VP70. As such, the caspase inhibitor z-VAD-mk

substantially inhibits viral processing to VP70. Virions can beormed by VP90, although they are not stable or inectious andin host cells the cleavage o VP90 to VP70 is critical or releaserom the cell. While cell death is not particularly associated withastrovirus inection, the characteristic blunting o villus tipscould be explained through a loss o cells. This villus bluntingalong with changes in barrier unction are the primary meansthrough which astrovirus causes diarrhea, although changes inion transport have not yet been studied.

Parasitic diarrhea. Diarrhea caused by parasites is unlike thato either bacterial or viral inections. For example, Giardia has aslow onset o diarrhea and can be present or months, while mostbacterial and viral inections are limited to 12 weeks.40 In addi-

tion, parasites are eukaryotic, which makes them larger and morecomplex than either viruses or bacteria and also more difcultto eradicate due to their similarity to the host. In act, it is notunusual or parasites to make their own versions o host proteins:For example serotonin and PGE

2are both made byEntamoeba

histolytica. The advanced mechanisms used by the two importantprotozoan parasites, Giardia lambliaand E. histolytica, to causediarrhea will be discussed below.

Giardia lamblia.Giardia lamblia(syn G. duodenalisand G.intestinalis) is the most common parasite o the human smallbowel and causes waterborne diarrheal disease worldwide.


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post inection.146 This decrease in TER was associated with anincrease in mannitol ux and can not be replicated with solubleactors or sonicated trophozoites. Inection does not alter levelso occuldin or its localization but does cause a decrease in levels

o ZO-1 without obvious dissociation rom the tight junction.

146

While secreted cysteine proteases seem a likely cause o this dropin TER, typical protease inhibitors such as E-64 or aprotinindid not block the drop in resistance. However, inactivation ocysteine proteases has been shown to block the later drop in resis-tance associated with hole ormation in monolayers.147

In addition to changes in tight junction activity, there arealso changes in ion transport. Analysis o amebic lysates sug-gests that there are two distinct actions on Cl - secretion: there isa Ca2+ dependent process, which is active in ileum and a cAMP-dependent process, which is active in colon (Fig. 9). The Ca2+-dependent response is only active on the serosal surace, suggestingthat these mechanisms are not activated until the epithelial layer

is breached.148

HPLC analysis and immunodetection ound thatamebic lysates actually contain low levels o serotonin and patho-genic strains contain twice the level o non-pathogenic strains.Serotonin is not the only host protein made by E. histolytica;recent studies show that prostaglandin E

2(PGE

2) is also made

when the amoebae are supplied with arachidonic acid (AA)149 (Fig.9). They have their own cyclooxygenase (COX)-like enzyme,which is capable o converting AA to PGE

2but is not inhibited

by indomethacin as are human COX enzymes.149 However, thisappears to be only a small part o the PGE

2response because

indomethacin is capable o blocking PGE2

production in human

there is an initial ulceration o the intestinal epithelium due tothe expression o several cysteine proteases, which degrade theextracellular matrix, and due to amoebapores which cause celllysis139,140 (Fig. 9). This results in cell detachment, reducing the

absorptive surace o the intestine. In addition, the cysteine pro-teases cause degradation o several complement actors includingC3, C3a and C5a, as well as immunoglobulins, thus bluntingthe innate immune response. While E. histolytica inection isinammatory, the parasite is resistant to neutrophils due bothto complement degradation and a C59-like complement-inac-tivating adhesion on the surace o the parasite.141 In addition,PMNs can be killed or alternately, neutralized through inhibi-tion o cathepsin G, a serine protease, thus decreasing the activ-ity o PMNs in areas o inection.142,143 In addition, macrophagesare activated due to stimulation o endosomal TLR-9 by parasiteDNA as well as activation o TLR2 and 4 by lipopeptidophos-phoglycan.144,145 These inammatory cells are thought to enlarge

the initial ask-shaped ulcerations which are characteristic oE.histolyticainection. Thereore, a combination o parasite medi-ated destruction and subsequent inammation causes the ulcer-ation o the epithelium which reduces absorption and disruptsbarrier unction.

In addition to these morphological changes there are also directeects on barrier unction. Because o their ability to induce celldamage and make apparent holes in monolayers, work with E.histolyticaand TER must be done early in inection. Leroy et al.ound there was a drop in resistance as early as 1 hour post inec-tion, while monlayers began losing cells between 3 and 6 hours

Figure 9. Epithelial destruction and inammation caused by E. histolytica. E. histolytica induces a ask-shaped ulcer usually spanning several villi rather

than sal clls as shon h. Th pthlal l ay s dstoyd both du to pathogn factos, such as th po fomng amobapos and xtacllu-

lar matrix cleavage by cysteine proteases, as well as host inammatory factors. E. histolytica lass PGe2

and causs a host PGe2

spons that sults

in colonic Cl- secretion, IL-8 secretion and PMN recruitment. While PMNs can be neutralized and phagocytosed by the parasite and complement is de-

graded by cysteine proteases, the infection is still highly inammatory. Macrophages have been shown to be activated by parasite DNA through TLR9

o altnatly though lpophoshpopptdoglycan actaton of TLr 2 o 4. in addton to PGe2, E. histolytica also scts sotonn, hch s capabl of

activating Ca+ dependent Cl- scton n th small ntstn.


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(summarized in Table 1). This is urther evident rom the hugeversatility in mechanisms utilized by pathogenic strains oE. coli,thus contributing to the complexity o pathogenesis. Remarkably,studies in recent years have progressed to defne E. coli-secretedproteins (Esps) that specifcally decrease ion and water absorp-tion, in contrast to toxin producing strains, which stimulatesecretion. Furthermore, activation o the mucosal immune andenteric nervous system as well as cytoskeletal arrangements andloss o absorptive surace seems to augment the outcome o inec-tion (Table 1). Although, malabsorption seems to be a prominentactor in rotavirus and Giardia inections, much progress needsto be made on the molecular mechanisms and virulence actors

underlying ion transport modulations associated with these inec-tions. Further investigation into how pathogenic strains oE. colimodulate host physiological responses in vivo to cause diarrheais critical to completely defne the disease. Similarly, our under-standing o the pathogenic mechanisms o noroviruses and astro-viruses is inadequate and needs extensive research. Unravelingthe pathophysiological mechanisms o these pathogens may helpidentiy novel therapeutic targets not only or diarrhea associatedwith enteric inections but also or a variety o other gastrointes-tinal diarrheal disorders.

Acknowledgements

We would like to thank Pro. Pradeep Dudeja, Dr. Waddah

Alreai, Andrew W. Ween, Athanasia Koutsouris and Dr. SeiYoshida or critical reading o the manuscript.

intestinal xenograts in SCID mice.150 The eects o PGE2 aremultiold with an increase in both production o IL-8, a potentchemoattractant, and an increase in Cl- secretion.150,151 Secretoryeects in mouse colon are attributed to PGE

2synthesis with only

a minor Ca2+-dependent component (25%).151 Pretreatment ocells with PGE

2causes desensitization to amebic lysates and the

COX inhibitors indomethacin and piroxicam block the rise in Isc

almost entirely.151 This mechanism is dependent on the produc-tion o cAMP and can be blocked with piroxicam, suggesting aCFTR-dependent mechanism in contrast to the Ca2+-dependentmechanism ound in small intestine151 (Fig. 9). Both mechanismsare active on the basolateral side o cells, suggesting that this is

not an early event but instead depends on initial infltration othe epithelial layer by the parasite prior to activation. The overallpicture oE. histolyticainection depends both on parasite com-ponents, including cysteine proteases, amoebapores, serotoninand PGE

2as well as the host inammatory response with no

single component providing a complete answer.

Conclusion

The elucidation o the mechanisms underlying inectious diar-rhea has progressed remarkably over the last decade and willcontinue to advance. Pathogen-specifc virulence actors and sig-naling cascades aect wide-range o cellular unctions such as ion

secretion, absorption, barrier unction and membrane trafck-ing events that elicit luminal uid accumulation causing diarrhea

Table 1. An o of th data psntd n ths assssng th lat contbuton of ach phnotyp to daha fo ach pathogn

psntd

Pathogen Type Symptoms Duration InflammationIncreased

secretion

Tight

junctions

Decreased

absorption

Loss of

cells*

Vibrio cholerae gam (-) pofus aty daha 34 days + +++ +++ +++ -

Clostridium

difficilegam (+) daha + f 3 days+ +++ ++ +++ + ++

Shglla spcs gam (-) daha + f 57 days +++ + +++ ? +

Escherichia coli

(EPEC)gam (-) daha 13 ks + - +++ +++ -

rotaus dsrNAomtng + daha +

f38 days - + ++ +++ +

Noous (+) rNA omtng + daha 12 days + ++ +++ ? +++

Astous (+) rNAomtng + daha +

f14 days + ? ++ ? ++

Gada protozoan daha 26 ks - + +++ ++ +++

Entamoeba

histolyticaprotozoan daha

ks to

months++ ++ ++ ? +++

*Loss of clls ncluds shotnng of ll.


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