bacteria and host interactions in the gut epithelial

8/2/2019 Bacteria and Host Interactions in the Gut Epithelial

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36 nature chemical biology | VOL 8 | JANUARY 2012 | www.nature.com/naturechemicalbiology

REVIEW ARTICLEPublished online: 15 december 2011 | doi: 10.1038/nchembio.741

T

he intestinal epithelium unctions to gain nutrients, retainwater and electrolytes and orm an ecient barrier against

oreign antigens and microbes. Te intestinal mucosa not onlyis critical or intestinal homeostasis but also serves as an inectiousoothold or the microbiota and invading pathogens. Tereore, thegut epithelium uses multiple deense mechanisms against microbes,including the luminal microbiota, a mucus layer, epithelial integrity,epithelial cell turnover and innate or acquired immune responses1.Despite the presence o these deensive systems, pathogenic bacte-ria can invade spaces that are usually devoid o microbes. Indeed,enteric bacterial pathogens such as Salmonella spp., Shigella spp.,Vibrio cholerae, enteropathogenic Escherichia coli (EPEC), entero-hemorrhagic E. coli (EHEC) and Yersinia can eciently inect andmultiply within the gut mucosa. Tese pathogens use a virulence-associated protein (eector) injectisome, termed a type III secre-tion system (3SS), that allows the bacteria to subvert the luminal

microbiota, hijack host signaling pathways, modulate innateimmune responses and circumvent innate deense barriers in thegut. We discuss the microbiota and the mucosal barrier as well asthe epithelium itsel and highlight bacterial stratagems to circum-vent these barriers with particular emphasis on the roles o bacterialeector proteins (able 1).

V Bacterial pathogens have evolved highly sophisticated protein exportsystems that have been classied into seven types (types IVII).Many Gram-negative bacterial pathogens have a 3SS, a type IVsecretion system (4SS) or both. 3SSs are evolutionarily andstructurally related to agellar export systems, whereas 4SSs arerelated to bacterial conjugation systems that translocate DNA. Te

3SS consists o approximately 20 highly conserved proteins2

thatorm a multiprotein complex composed o the ollowing distinctiveparts: (i) a basal body, which is the channel spanning the bacterialmembraneperiplasm; (ii) a needle structure, which is the core 3SSprojection that spans the bacterial membranes and the extracellularspace; and (iii) a needle tip, which orchestrates the insertion o thetranslocon that links the needle to the host membrane (Fig. 1a).3SS assembly is a tightly regulated and ordered process in whichthe basal body is ormed in the inner and outer membranes beorethe needle structure is generated. When the needle is completely

b p

h a1, m ow1, m K2, h m1 & c skw1,2*

Te t s ts s e nst nvdes, wees esdent ens nd en nvdn te ntetntte wt te t epte nd nene te st e nd ne sstes. Te epte e seves s nnets td n te ptens nd s n ent pt ptens t dssente nt deepe tsses. Entete ptens n eent net te t s sn spstted vene enss tt w tet vent te deense es n te t. We pvde n vevew te pnents te s e nd dsss tete sttes tt vent tese es wt pt epss n te es te efet ptens.

ormed, the needle tip is secreted and the assembly o the apparatusis completed. Te 3SS is activated upon contact with host cells via

the needle tip; translocators are then secreted by the 3SS and createa pore in the host cell membrane that provides access to the host cellcytosol. Te temporal ordering o 3SS assembly and eector secre-tion is thereore essential or bacterial virulence (Fig. 1a).

Bacterial pathogenesis that uses 3SSs to deliver a subset o eec-tor proteins into host cells is dependent on 3SS activity. Consistentwith this notion, inactivating the 3SS can almost completely abol-ish pathogenesis2. Tereore, activating 3SS at the appropriate siteand time promotes successul bacterial inection. 3SS activity, aswell as eector production and secretion, is controlled by tran-scriptional regulators in response to the intestinal environment.For example, in Shigella, 3SS activity is responsive to variationsin oxygen concentrations3 (Fig. 1b). Oxygen gradients are presentbetween the intestinal tissues and anaerobic intestinal lumen, and

Shigella in the lumen sense environmental oxygen through an uni-dentied O2 sensor and transduce signals to Fnr, a global regulatorthat modulates anaerobic metabolism. Fnr represses the transcrip-tion o spa32 and spa33, whose products regulate 3SS needlelength and eector secretion, respectively. Tereore, Fnr preventsthe secretion o eector proteins and primes the extension o the3SS needle until the bacteria directly contact the target epithelialcells. When Shigella reach a relatively oxygenized area above theintestinal epithelial cells, Fnr-dependent gene repression is silenced,the 3SS is activated and invasion proceeds3.

3SS activity in Salmonella enterica var. yphimurium(S. yphimurium) also responds to the environmental pH4,5 (Fig. 1c).Salmonella inect the intestinal mucosa using two distinct andsequential pathways: the Salmonella pathogenicity island 1 (SPI-1)-

dependent pathway, which is involved in bacterial invasion, and theSPI-2dependent pathway, which is involved in bacterial intracel-lular survival. Salmonella inection o the gut triggers inammationdue to the delivery o eectors via 3SS-1 and 3SS-2 encoded bySPI-1 and SPI-2, respectively6. Salmonella enter intestinal epithe-lial cells and phagocytic cells where they survive and replicate inmaturing Salmonella-containing vacuoles (SCVs), compartmentsrequired or bacterial replication, intracellular survival and systemicinection6. Once Salmonella invade and replicate within host cells,oll-like receptors (LRs) in the host recognize pathogen-associated

1Department o Microbiology and Immunology, Institute o Medical Science, University o Tokyo, Tokyo, Japan. 2Department o Inectious Disease Control,

International Research Center or Inectious Disease, Institute o Medical Science, University o Tokyo, Tokyo, Japan. *e-mail: [email protected]


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nature chemical biology | VOL 8 | JANUARY 2012 | www.nature.com/naturechemicalbiology 37

REVIEW ARTICLENaTurE chEmical biologydoi: 10.1038/nchembio.741

molecular patterns (PAMPs), which activate the innate immuneresponse to limit and clear the inection. However, Salmonellasense the acidication o the SCVs, which occurs as a result o LR-mediated innate immune responses, and use this host response asa cue to induce the expression o bacterial virulence genes encod-ing the 3SS-2 component and to assemble it4 (Fig. 1c). Although3SS-2 assembly is primed within the SCVs, eector secretion isprevented by a regulatory complex, which is most likely a ternarycomplex ormed within the bacterial cytosol under acidic condi-tions. Once the translocator at the tip o 3SS-2 orms a membranepore, 3SS-2 senses the neutral pH outside the SCVs (Fig. 1c). Anunidentied pH sensor, which senses an elevated pH, transduces a

dissociation signal to the regulatory complex, thereby triggering thedelivery o eector proteins into and across the vacuolar membrane,resulting in prolonged bacterial survival and replication5 (Fig. 1c).

Because environmentally responsive regulators (responding totemperature, O2, pH, bile salts or ions) o 3SS activity are highlyconserved across many Gram-negative bacterial pathogens, viru-lence in the gut is believed to be regulated by similar mechanisms.

m j

Te mammalian intestine contains approximately 1014 commensalbacteria, representing approximately 1,000 species. Tese lumi-nal commensal bacteria are a predominant part o the microbiota(hereafer we reer to commensal bacteria as the microbiota), andthey contribute to intestinal digestive unction, inuence epithelialmetabolism and stimulate both epithelial cell prolieration and gutimmunity as well as competing with enteric pathogens79. Tus,these bacteria can directly or indirectly promote resistance to path-ogenic bacterial colonization.

Microbiota limit bacterial colonization and stimulate epithelialturnover, which occurs at a two-old aster rate in conventional micethan in germ-ree mice10. Comparing the transcriptional proles othe intestinal epithelia o germ-ree and conventional piglets simi-larly shows that conventional epithelia expresses proteins that con-

tribute to epithelial turnover and are involved in the biosynthesis omucin, an important component o the gut mucosal barrier, and inimmune system priming. Both o these outputs are important orresistance to pathogen colonization11.

Short-chain atty acids (SCFAs), such as acetate, propionate, or-mate or butyrate, are produced as end metabolites by the micro-biota and prooundly inuence gut barrier unction, host immunity,epithelial prolieration and bacterial pathogenesis9 (Fig. 2a).Bidobacteria species that produce high concentrations o ace-tate as an end product o carbohydrate metabolism can preventEHEC inection and the release o Shiga toxin, a crucial actor inlethal inection, rom the lumen to the blood stream o the host 12.Examining the role o microbiota in chemically induced colitis ingerm-ree mice containing selective gut microbiota (called gnoto-

biotic mice) showed that acetate production by commensal bacteriaor acetate administration stimulated the G proteincoupled recep-tor 43 (GPR43), an acetate chemoattractant receptor on immunecells that regulates inammatory responses and allows the gnotobi-otic mice to eciently recover rom colitis13.

Likewise, butyrate, produced mainly by Fecalibacterium praus-nitzii, Eubacterium rectale and Roseburia species, prevents bacterialinection by directly aecting virulence gene expression, upregu-lating the expression o the epithelial antimicrobial peptide LL-37and providing energy or the colonic epithelium, which in turnallows epithelial prolieration and injury repair14. For instance, rab-bits that were pretreated with butyrate and inected with Shigellahad reduced colonic inammation and bacterial loads in the stoolbecause butyrate upregulates the expression o the Cap18 gene,

which encodes the rabbit homolog o LL-37 and enhances bacteri-cidal activity15.Microbiota contribute to the development and unction o the

immune system. In the intestines o germ-ree mice, Peyers patches,which are aggregated lymphatic ollicles, are poorly developed. Inaddition, germ-ree mice have altered compositions o CD4+ cellsand IgA-producing B cells in the lamina propria, the underlying tis-sue o epithelial mucosa, compared to conventional mice and rats16.Te induction o -lymphocyte subsets is augmented by distinctivespecies o the luminal microbiota. Segmented lamentous bacteria(SFB), or instance, adhere to the luminal surace o Peyers patchesin the mouse intestine and strongly stimulate the dierentiation o helper 17 (H17) cells in the lamina propria, which contribute toresistance against colonization by pathogens17,18 (Fig. 2b). Althoughthe precise mechanism o SFB-promoted

H

17 dierentiation

O2

Low

High

Epithelial cells

Lumen

Basolateral

SCV

Acidification

1. Assemble T3SS

Acid pH

3. Sense neutral pH

Neutral pH

4. Secrete effector

Eector

TLRs

Inner membrane

Outer membrane

Periplasm

Host cell membrane

Shigella

Transcription

Fnr

Low oxygen

TranscriptionFnr

High oxygen

Eector

Needle

Base

a

b

Eector

Eector

Needletip

Translocator

Type III secretion system

PAMPs

Spa32 Spa33

Spa32 Spa33Eector secretion

No eector secretion

c

Virulencegene

2. Block effector secretion

Regulatorycomplex

Eector

Disassemblyregulatorycomplex

Eector Eector

Pore

Eector

Fe 1| T3SS s deve sste te efets. () The T3SS

is composed o a basal body, a needle structure and a needle tip. When

the needle structure is complete, the needle tip protein is secreted. Once

bacteria contact host cells via the needle tip and activate the T3SS, two

translocator proteins are secreted via the T3SS and orm a pore in the host

cell membrane. The eector proteins can pass through the needle and

translocation pore into the host cell cytosol. () Shigella modulate T3SS

activity by sensing environmental O2 conditions. Under the anaerobic

conditions o the intestinal lumen, Fnr prevents the secretion o T3SS

eectors by repressing the transcription o spa32 and spa33.

() Salmonella use pH as a signal or T3SS-2 assembly and eector

secretion within SCVs. Salmonella sense TLR-induced acidication within

SCVs and induce the expression o the virulence gene locus SPI-2 as well

as T3SS-2 assembly. Ater the T3SS-2 is assembled, Salmonella sense

the neutral pH outside o the SCVs and induce the disassembly o the

regulatory complex, thereby triggering eector secretion.


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REVIEW ARTICLE NaTurE chEmical biologydoi: 10.1038/nchembio.741

remains partly unclear, SFB adherence to Peyers patches seems toinduce serum amyloid A, an apolipoprotein produced by the epithe-lial cells in Peyers patches that inuences cell adhesion, migrationand prolieration. In another example, the colonization o germ-reemice with a dened mix oClostridium strains revealed that someClostridium species stimulate the accumulation o Foxp3+ regula-tory (reg) cells in the colonic lamina propria, resulting in an amelio-ration o colitis in chemically induced mouse models o the disease19(Fig. 2b). Te Clostridium species orm a thick bacterial colonizing

layer on the epithelium, where they stimulate the production o theactive orm o GF-b, which induces the dierentiation o naive cells into reg cells

19 (Fig. 2b).Te impact o the microbiota on colonization resistance is ur-

ther illustrated in an antibiotic-treated mouse model o inection,which has been traditionally used to study and characterize bacte-rial pathogenesis20. Although the composition o the microbiotain the small intestines or colon o untreated mice is markedly di-erent, streptomycin treatment substantially decreases the luminalconcentrations o SCFAs21. Formate, which is present at higherconcentrations in the small intestine than in the colon, leads tothe upregulation o invasion genes in Salmonella. By contrast,butyrate, which is present at higher concentrations in the colonthan the small intestine, leads to the downregulation o invasion

genes, indicating that SCFA concentrations aect the preerentialinection o the small intestine bySalmonella9,21,22.Until recently, it was poorly understood how enteropathogenic

bacteria circumvent colonization resistance mechanisms. Gutinammation triggered by enteric pathogens disrupts the micro-biota, allowing pathogens to outgrow the luminal microbiota.Studies with acute Citrobacter rodentium or S. yphimuriuminected mouse models demonstrated that virulent bacteria canoutgrow the microbiota by delivering 3SS eectors to invadeand multiply within host cells that trigger gut inammation7,23,24.S. yphimurium promotes its own outgrowth over the gutmicrobiota using an interesting mechanism. During inection,S. yphimurium induces inammation that is accompanied bythe production o reactive oxygen species and nitric oxide radi-cals. Tese species can react with luminal thiosulate (S

2

O3

2), a

compound produced to detoxiy the hydrogen sulde (H2S)produced by colonic bacteria, to orm tetrathionate (S4O6

2).Salmonellas ability to use tetrathionate as a terminal electronacceptor during respiration gives S. typhimurium a growth advan-tage over microbiota in the inamed gut25 (Fig. 2c).

Microbiota help exclude pathogens by preventing colonizationthrough enhancement o intestinal immune development and theproduction o SCFAs and bactericidal proteins. Nevertheless, somebacterial pathogens can circumvent these resistance mechanisms

by delivering eectors and toxins that alter the microbiota compo-sition and thus allow pathogenic bacteria to successully colonizethe intestinal epithelium. Functional crosstalk among the luminalmicrobiota, intestinal immune molecules and invading pathogensis paramount in determining the conditions in the gut.

m j p Te gut epithelium is covered by a thick mucus layer that acts asa rontline deense barrier against the microbiota and pathogenicbacteria26,27. However, enteric bacterial pathogens have evolvedmechanisms to circumvent this mucus barrier and directly accessthe epithelial surace. Te mucus layer is largely composed omucin, which contains various digestive enzymes and antimicro-bial peptides as well as immunoglobulins (Fig. 3a) and contains two

sublayers: an outer loose mucus layer and an inner rmly adherentmucus layer. In the inner layer, the epithelial cell proximal suraceis covered with glycocalyx, which consists o membrane-boundmucin glycoproteins. Mucins are produced and secreted by gobletcells throughout the intestinal tract, and they help remove the gutcontents and intruding microbes. Epithelial cells and Paneth cellssecrete antimicrobial peptides that help prevent bacteria rom pen-etrating the inner mucus layer. Bacterial colonization is thereorelimited to the outer loose mucus layer, where the bacteria interactwith the oligosaccharides o secreted mucin glycoproteins, whereasthe inner layer is devoid o bacteria26,27 (Fig. 3a).

Although mucin is constitutively secreted at a basal level, mucinsecretion can change in response to luminal conditions or bacte-rial inection. Mucin secretion by goblet cells is regulated by a vari-ety o secretagogues, including microbial products, inammatory

Fe 2 | intetn etween te t nd t ptens. () SCFAs produced as end metabolites by the microbiota are important elements

that prevent the colonization o bacterial pathogens. () The epithelial cell layer separates the gut lumen rom the lamina propria, the tissue beneath the

mucosal epithelium that contains various myeloid and lymphoid cells. The lamina propria contains a large number o immune cells and acts as an eector

site or IgA production and T-cell responses. Luminal antigens are transported through M cells to the subepithelial dome o Peyers patches, which contain

dendritic cells, T cells and B cells. SFB adhere to the luminal surace o Peyers patches, and TH17 cells dierentiate in the lamina propria. Some Clostridium

species promote the release o the active orm o TGF-b, which promotes Treg cell dierentiation and accumulation in the colonic lamina propria, therebypreventing infammatory responses. () S. Typhimurium induces infammation that causes the production o reactive oxygen species that react with

luminal thiosulphate to orm tetrathionate. Salmonella can use tetrathionate as a terminal respiratory electron acceptor.

Neutrophil

Inflammation

Salmonella

Microbiota

ROS

Thiosulfate Tetrathionate

Salmonellagrowth

Epithelial cells

H2S

c

SFB

TH17 cell

accumulation

Clostridium

Treg

cellsNaive

b

Lumen

Lamina propria

Epithelial cellsM cells

: Macrophage

: Dendritic cells

: T cells

: B cells

Peyers patch

Inflammation

Adherence

Serumamyloid A

?

TGF-

Microbiota

Bacterial pathogens

Epithelial

cells

IgA

Antimicrobialpeptide

Epithelial cells

Lumen

Basolateral

Bifidobacterirum

Acetate

EHECStx2

Microbiota

Immune cells

GPR43

Chemoattractant

ButyrateAntimicrobialpeptides

a

Shigella

Microbiota

Acetate

GPR43

Inflammation


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as Pic, a serine protease that degrades mucin (able 1)35; StcE, a zincmetalloprotease that cleaves mucin-type O-glycosylated proteins(able 1)36; Hap, a zinc metalloprotease; agA, a Hap homolog withmetalloprotease activity that is distinct rom Hap (able 1)37,38; ormucin-degrading enzymes39, which degrade mucin oligosaccharidesand reduce mucus viscosity and the release o antimicrobial pep-tides. Tus, although the mucus layer prevents bacterial pathogensrom accessing and breaching the epithelial lining, pathogens havedeveloped specic mechanisms to penetrate the mucus barrier.

ep

Cell-cell and cellbasement membrane interactions in the epithe-lium orm a barrier that prevents bacteria rom translocating tothe subepithelial layer. Epithelial cell-cell adherence is sustained bytight junctions (apical multiprotein complexes that orm a selec-tively permeable seal between cells), adherence junctions (junc-tions subjacent to tight junctions that orm a strong interaction withjunctional molecules between cells), gap junctions (paired connexinhemichannels) and desmosomes (adhesive junctions between cells).Te apical junctions, composed o tight junctions and adherencejunctions, consist o transmembrane and cytoplasmic scaoldingproteins that associate with actin laments and regulate epithelialparacellular permeability40,41.

ight junctions are composed o zonula occludens (ZO-1 andZO-2) and junctional adhesion molecules (JAM-1, claudin and

occludin), and they unctionally segregate the apically expressedmembrane proteins rom those expressed on the basolateral mem-brane on polarized epithelial cells (Fig. 4a). However, tight junctionsare highly dynamic structures, and their permeability is regulated byseveral physiological and pathophysiological conditions. For exam-ple, inammatory cytokines can disrupt tight junctions and impairgut barrier integrity. reating epithelial monolayers with NF-aor IL-1b increases tight junction permeability by stimulating tran-scription and activation o myosin light chain kinase (MLCK)4244.Te phosphorylation o MLC by MLCK stimulates perijunctionalactomyosin contraction, leading to the distension o transmem-brane tight junction strands and increased paracellular permeabil-ity45. Tereore, the breakdown o tight junctions during bacterialinection results in gut barrier ailure, ofen termed leaky gut, whichsubsequently acilitates the translocation o bacteria and the luminal

mediators, hormones, signaling mediators, growth actors and inec-tious bacteria26,27. Mucin expression can also be controlled at thetranscriptional level through several mechanisms. For example, therecognition o PAMPs via LRs and NOD-like receptors activates thedownstream inammatory signaling pathways that induce the tran-scription o mucin genes26,27. During Pseudomonas aeruginosa inec-tion o the colonic epithelium, lipopolysaccharide (LPS) inducesMuc2 gene transcription by activating a LR4-dependent pathway28(Fig. 3b). In contrast, inection with the Gram-positive bacteriaStaphylococcus aureus induces the transcription o the Muc2 genethrough a LR-independent pathway29. Te bacterial lipoteichoic

acid activates platelet-activating actor receptor, which is a G pro-tein-coupled receptor, and transduces signals to the epidermalgrowth actor receptor (EGFR) via ADAM10 metalloproteinaseactivity. Activation o this pathway, in turn, stimulates the Ras-MAPK-NF-kB pathway and activates mucin gene transcription29(Fig. 3b).

Te mucus layer acts as a protective barrier against colonization.For instance, inoculation oMuc1-/- mice (Muc1 encodes a cell-surace mucin) but not wild-type mice with Campylobacter jejuniresults in rapid systemic inection30. Similarly, approximately ve-oldmore H. pylori colonizeMuc1-/- micethan colonize wild-type mice.Wild-type mice develop only mild gastritis afer H. pylori inectionor 2 months, whereasMuc1-/- mice develop severe atrophic gastri-tis and lose gastric parietal cells31. Intriguingly, gastric epithelial cells

can shed Muc1 in response to H. pylori inection, and the releasedMuc1 acts as a decoy that binds pathogens, preventing bacterialadhesion to the epithelial surace32 (Fig. 3b). Tus, the interaction opathogens with mucin can contribute to pathogen elimination due torapid mucin secretion and mucus shedding. Cell-surace mucins canlimit the colonization o bacterial pathogens.

Enteric bacterial pathogens have evolved mechanisms to senseand circumvent the mucus barrier and reach the epithelial cell sur-ace. Although the mechanism remains unclear, C. jejuni uses Muc2as an environmental cue to modulate the expression o genes involvedin colonization and pathogenesis33. Te agella and chemotaxis sys-tems are used by many enteric pathogens to traverse the mucus layerand access the epithelial cell surace. Disrupting agella unctionreduces pathogenicity, highlighting the ability o bacterial motility topromote inection34. Some enteric pathogens deploy enzymes, such

Ras

MAPK

NF-B

Muc2

P. aeruginosa

Mucus layer Mucus-degradatingenzyme

Pathogens

Inner mucus layer

Outer mucus layer

a b

Intestine

Microbiota

MUC

MUC MUC MUC

Goblet cell Goblet cell

Antimicrobialpeptides

Immunoglobulin

Bacterial pathogens

MUC1

Lamina propria

Lumen

GproteinADAM

10

TLR4

LPS

PAFREGFR

LTA

Src

H. pylori

S. aureus

Cell-surfacemucin

O-glycosylation

Intestine

MUC

Secretedmucin

Bacterialpathogens

MUC

Fe 3 | Te s e s t e. () The mucus layer serves as a rontline deense against intruding bacterial pathogens. () Pathogenic

bacterial inection causes Muc2 production through TLR-dependent or TLR-independent NF-kB pathways, whereas some bacterial pathogens have

mucolytic enzyme activities that destroy these mucus layers. PAFR, platelet-activating actor receptor.


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contents across the damaged epithelial lining and urther promotesbarrier damage and disease progression.

Bacterial pathogens use tight junctions to acilitate their interac-tions with the epithelium and penetrate into deeper tissues. Becausetight junction components interact with the actin cytoskeleton tosustain the integrity o cell-cell adherence, gastrointestinal pathogenssubvert several signal pathways that regulate tight junctions such asthe protein kinase C pathway, which regulates actin laments, andthe Rho GPase pathway, which modulates actin cytoskeleton rear-

rangement1,46 (Fig. 4b).3SS eectors or toxins rom enteric pathogens can subvert

host signal pathways that regulate actin organization and tightjunctions. Some eectors directly modulate Rho GPases byacting as guanine exchange actors (GEFs), which convert inac-tive GDP-bound Rho GPases to the active GP-bound ormsand disrupt tight junction structure and unction. Because thecomponents o tight junctions are constantly recycled betweenthe plasma membrane and cytosol, regulating tight junctioncomponents is important to maintain epithelial integrity. othis end, Rho GPases play pivotal roles: RhoA triggers acto-myosin assembly and contraction through Rho-associated kinase(ROCK)-mediated MLC phosphorylation, and Rac1 and Cdc42activate the PAK-LIMK-colin pathway, which leads to actin

lament stabilization and tight junction modication (Fig. 4b).Because signaling o Rho GPases is tightly interconnected, theactivation or inactivation o one Rho GPase can cause an imbal-ance in other networks, resulting in actin disorganization andtight junction disruption41. For example, EPEC disrupts tightjunctions by altering actin cytoskeleton remodeling through the3SS eectors EspM, Map and NleA (Fig. 4c and able 1)47. Map

activates Cdc42, whereas EspM activates RhoA through its GEFactivity and alters the localization o tight junctions4851. NleAbinds to and intereres with COPII-dependent protein track-ing, thus altering tight junction components and permeability52.C. rodentium inection o murine intestinal epithelial cells resultsin diminished barrier unction associated with disrupted tightjunctions53. C. rodentium secretes a lymphocyte inhibitory actorcalled lymphostatin, which can upregulate RhoA and downregu-late Cdc42. When mice were inected with C. rodentium lacking

the liA genes, which encode lymphostatin, the intestinal barriermediated by tight junctions was not compromised54.

Te gastrointestinal pathogen Vibrio parahaemolyticus deliv-ers one eector that induces cell rounding, VopS (able 1), viathe 3SS55. VopS catalyzes the AMPylationthe transer o AMProm AP to the threonine residueso proteins such as Rac1,Cdc42 and RhoA via a phosphodiester bond56, thereby blockingRho GPase signaling and reducing actin cytoskeleton remode-ling. Tis ultimately disrupts the integrity o the actin cytoskeletonin epithelial cells56 (Fig. 4d).

S. yphimurium also delivers eectors that disrupt tight junc-tions5759 (Fig. 4e). SopB indirectly activates Rho GPases, whereasSopE and SopE2 act as GEFs that activate Rac1 and Cdc42 (re. 6).Activation o Rho GPases by the S. yphimurium eectors acili-

tate the uptake o bacteria by epithelial cells, which also aectstight junction structure and unction. ight junction disruption byS. yphimurium can be blocked with an inhibitor o geranylgera-nylation, a unction required or Rho GPase activation, indicatingthat Rho GPase activation by these eectors is a cue or tight junc-tion disruption57. S. yphimurium SopE-mediated intestinal inam-mation is abrogated in Casp-1/, Il1r1/ or Il18/ mice, indicating

Te 1 | Seeted expes te ptens nd t deense sste

bte sttes bte Ft(s) Fntn hst tet(s) reeene(s)

Disruption o

mucus layer

Shigella, EAEC Pic Degradation o mucin Mucin 35

EHEC StcE Degradation o mucin Mucin 36

V. cholerae Hap, TagA Degradation o mucin Mucin 37,38

Breach cell-cell

junction

H. pylori CagA Disrupts cell-cell junctions and cell polarity ZO-1, PAR1 6163

VacA Disrupts cell-cell junctions Unknown 64

Urease Disregulates TJs MLCK, ROCK 65

Unknown Disrupts TJs IL-1R1 66

EPEC Map Disrupts TJs Cdc42 48

EspM Disrupts TJs RhoA 4951

NleA Disregulates TJs COPII 52

V. parahaemolyticus VopS Disrupts cytoskeletal integrity Rho GTPase 56

Salmonella SipA, SopB, SopE, SopE2 Disrupt tight junctions Rho GTPase 57

Epithelial cell death

and shedding

Shigella Unknown Induces mitochondrial dysunction BNIP3, CypD 72

Unknown Inhibits mitochondrial dysunction Bcl2 72

Salmonella AvrA Inhibits cell death MAPKK 75,76

SopB Inhibits cell death PI3K/AKT 77,78

EPEC EspF Induces apoptosis Abc2 81

H. pylori VacA Induces apoptosis p38, Bax 82

Regulation o epithelial

cell turnover

H. pylori CagA Increases prolieration Cyclin D1 89

Inhibits apoptosis and prolieration MCL-1 87

C. rodentium Unknown Increases prolieration b-catenin, CKI 86

EPEC Ci Arrests cell cycle NEDD8 91

NleH Inhibits apoptosis and prolieration BI-1 96

Shigella IpaB Arrests cell cycle Mad2L2 90

TJ, tight junctions.


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modulates MAPPKs and inhibits the JNK signaling pathway, helpsdampen inammatory and cell death responses75,76 (Fig. 5b). SopB,an inositol phosphatase, activates the serine-threonine kinase Akt,which plays a critical role in cell survival and prolieration, allowingSalmonella to counteract epithelial cell apoptosis77,78.

EPEC delivers a subset o eectors (able 1) via the 3SS, andthese eectors have an impact on a variety o host cell-signaling

pathways that are involved in cytoskeletal rearrangement, disrup-tion o epithelial intercellular junctions, epithelial cell death andimmune modulation47. EspF disrupts the mitochondrial mem-brane potential, resulting in cytochrome c release and apoptosis79,80.EspF interacts with Abc2, an antiapoptosis actor, and reduces itsexpression within the mitochondria, thereby inducing apoptosis81.H. pylori colonization causes apoptosis o gastric pit cells byinducing oxidative stress, in which p38 activation and Bax oli-gomerization have a key role. VacA-mediated translocation o Baxoligomers into the mitochondria in response to H. pylori inec-tion results in decreased mitochondrial membrane potential andapoptosis82.

Epithelial cell death at the early stage o inection is predomi-nantly a consequence o host deense mechanisms. However, some

pathogens elicit epithelial cell death, whereby they breach the epi-thelial barrier, access the underlying tissues and obtain nutrients.Afer the bacteria ully propagate within epithelial cells, theirdeath acilitates bacterial egress and spread to other host tissues.

ep vTe gut epithelium undergoes continuous sel-renewal to maintaintissue homeostasis and eliminate damaged cells. Tere is a balancebetween the elimination o damaged cells and the generation o newcells rom a stem cell population in the intestinal crypt ( Fig. 5a).Aside rom physiological renewal, epithelial turnover can accelerate(or decelerate) in response to changes in the luminal environment.Epithelial turnover o the Drosophila melanogastergut accelerates inresponse to inection with Erwinia carotovora, Pseudomonas spp. orSerratia marcescens83,84. In mice, C. rodentium stimulates b-catenin

signaling via upregulating casein kinase I (CKI) production. Asa result, C. rodentium stimulates the prolieration o cryptic stemcells, leading to hyperplasia and increased crypt length85. Tesestudies show that increased epithelial cell turnover in response tobacterial pathogens is a host deense mechanism that eliminatesinected cells, limits persistent bacterial colonization and maintainstissue homeostasis.

Some bacterial pathogens, such as H. pylori, Shigella or EPEC,counteract rapid epithelial turnover and maintain epithelial cellsas a replicative niche. Colonization o the gastric supercial epi-thelium by H. pylori causes an imbalance between epithelial cellprolieration and apoptosis86. H. pylori CagA hijacks multiple cellsignaling pathways to promote persistent colonization o the gas-tric epithelium by activating transcription actors such as nuclearactor o activated cells, serum response actor, NF-kB or -cellactorlymphoid enhancer actor (CF-LEF)60,87. CagA-mediatedupregulation o these transcription actors results in the produc-tion o cyclin D1, a cell-cycle regulator and the enhanced survivalo epithelial cells88. H. pylori can attenuate apoptosis o maturatedgastric pit cells, which are normally shed every 23 days in a gerbilmodel o inection86. When epithelial cell apoptosis was stimulated

with etoposide, H. pylori inection augmented epithelial expres-sion o the prosurvival actor Erk and the antiapoptotic proteinMcl-1 in a CagA-dependent manner, enhancing the bacterial loadin the gerbil stomach86 (Fig. 5b). Shigella can manipulate epithe-lial cell turnover by directly accessing cryptic epithelial progeni-tors; specically, the bacteria deliver the IpaB eector (able 1)via a 3SS into progenitors. In HeLa cells and rabbit ileal-loopinection models, IpaB was shown to negatively regulate cell-cycleprogression by directly binding to Mad2L2, an anaphase-promotingcomplex inhibitor, to promote bacterial colonization89.

Bacterial pathogens can also manipulate neddylation (attach-ment o the ubiquitin-like protein NEDD8) to regulate epithelialturnover and promote inection9092. Some EPEC strains deliver Ci(able 1), an eector with a papain-like hydrolytic old that includesa cysteine-histidine-glutamine catalytic triad, which blocks the cell

H. pylori

CagA

ERK

Proliferationand turnover

Apoptosis

MCL1

a b

Cif

Cullin-RINGubiquitin ligase

NEDD8

Gln40Glu40

Cell-cycle arrest

deamidation

EPEC

Crypt

Epithelial cell

Villus

Progenitorcell

Panethcell

Cytochrome c

Shedding cell

Migration

Dierentiation

JNK

MAPKK

Ac

Ac

Casp-3

MAPKK

AvrA

Salmonella

Stemcell

Lamina propria

Fe 5| bte nteeses nst epte e det nd tnve. () The gut epithelium undergoes continuous sel-renewal to maintaintissue homeostasis and eliminate damaged cells. The epithelial cells that line the gastrointestinal lumen are constantly renewed through a process in

which stem cells generated in the crypts migrate to the tip o the villi and ultimately peel o into the lumen. () Epithelial cell death and turnover are highly

dynamic responses to bacterial inection and limit persistent bacterial colonization, whereas some bacterial pathogens deploy countermeasures against

rapid epithelial turnover to prolong their survival. Selected eectors are shown.


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cycle by causing both G1-S and G2-M arrest93,94. Ci selectively bindsto NEDD8 and deamidates Gln40, inhibiting the activity o theNEDD8-conjugated Cullin-RING E3 ubiquitin ligases90 (Fig. 5b).Inhibition o these enzymes may induce ormation o actin stressbers and contribute to cell-cycle arrest90.

During EPEC inection, the eector NleH (able 1) inhibits apo-ptosis by binding to the endoplasmic reticulum six-transmembraneprotein Bax inhibitor (BI-1). Independent o its kinase activity, NleHinhibits several cellular responses associated with apoptosis, includ-

ing elevation o cytoplasmic Ca2+ concentrations, nuclear conden-sation and activation o caspase-3 (re. 95). In a mouse model oC. rodentium inection, NleH inhibited procaspase-3 cleavage atC. rodentium colonization sites in the intestine95. Although the exactrole o NleH in EPEC inection remains partly speculative, it islikely that NleH acilitates EPEC pathogenesis by reducing entero-cyte loss, which sustains the replicative niche.

Tese ndings suggest that epithelial turnover changes inresponse to bacterial inection to limit persistent bacterial coloni-zation. Enteric pathogens can deploy countermeasures to preventrapid turnover o epithelial cells in order to maintain epithelial cellsas a replicative niche.

c ppv

Mutualism between the gut mucosa and microbiota is the mostimportant actor in sustaining gut homeostasis. Enteric bacterialpathogens have highly evolved mechanisms to shif the balanceo host-microbiota mutualism or the pathogens benet. Manyenteric Gram-negative pathogens deploy 3SS eectors that pro-mote the survival and colonization o these pathogens within thegut by disrupting microbiota-mediated colonization resistance,circumventing the innate barriers o the gut and modulatingmucosal innate immune responses. 3SS eectors are bacterialexecutioners that can directly or indirectly modiy the target hostproteins and subvert host cellular and immune unctions. Tedevelopment o various new technologies, bioinormatics, ani-mal and other eukaryotic model systems will help elucidate theunidentied mechanisms o eector proteins and provide insights

that can be used to develop new antimicrobial drugs and thera-peutics as alternatives to antibiotics. Antibiotics have been exten-sively used to selectively target bacterial pathogens to treat andprevent many inectious diseases96,97. However, antibiotics also dis-rupt the composition o the microbiota, ofen leaving an imprinton the composition o the microbiota afer the antibiotic treat-ment has been discontinued, thereby promoting the emergence oantibiotic-resistant bacteria. Tus, we need to generate new drugsthat target bacterial pathogenesis rather than kill pathogenic bac-teria96,97. In this regard, 3SS inhibitors have been recently con-sidered new drugs, and several small-molecule compounds (bothsynthetic compounds and natural products) have been identiedas 3SS inhibitors. For example, a group o salicylidene acylhy-drazides inhibits the 3SS activity o many bacterial pathogens by

blocking the assembly o the 3SS needle complex, thereby pre-venting 3SS eector secretion and virulence98100.New insights are providing a blueprint or the dynamic net-

works among the commensal (and pathogenic) microbes andthe gut immune system in condition o both health and disease.In recent years, there has been an explosion o new analyticaltools that help to identiy the microbial and host actors aect-ing the mutualistic relationship between the microbes and gut.One relatively new approach, systems biology, allows us to com-prehensively understand how the gut commensal (and patho-genic) bacteria establish replicative niches and how the hostimmune system responds to bacterial inection. We envision thatan understanding o the mechanisms by which bacteria disruptthe mutualism between the mucosa and microbiota as well aspromote inection o the gut epithelium will provide avenues to

develop new therapeutics that control microbial inection, exces-sive host inammatory responses, microbiota composition andgut homeostasis.

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akwTis work was supported by Grant-in-Aid or Specially Promoted Research (23000012

to C.S.); a Grant-in-Aid or Scientic Research (S) (20229006 to C.S.); a Grant-in-Aid

or Young Scientists (A) (23689027 to M.K.); a Grant-in-Aid or Young Scientists (B)

(23790471 to M.O. and 23790472 to H.A.), a Grant-in-Aid or Scientic Research (B)(23390102 to H.M.); a Grant-in-Aid or Challenging Exploratory Research (23659220 to

H.M.); and a grant romthe Japan Initiative or Global Research Network on Inectious

Diseases to C.S. rom the Ministry o Education, Culture, Sports, Science and echnology.

Part o this work was supported by grants rom the Naito Foundation (to H.A.).

cp f Te authors declare no competing nancial interests.

a

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