do bacterial genotoxins contribute to chronic inflammation, genomic instability and tumor...

REVIEW ARTICLE

Do bacterial genotoxins contribute to chronicinflammation, genomic instability and tumor progression?Lina Guerra, Riccardo Guidi and Teresa Frisan

Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden

Introduction

Epidemiological evidence has linked chronic bacterial

infections with an increased risk of tumor development.

Heleicobacter pylori is associated with gastric cancers

and has been classified as a type I carcinogen by the

World Health Organization [1]. Other known associa-

tions between bacterial infection and human cancers are

Salmonella enterica serovar Typhi and carcinoma of the

gallbladder in chronic carriers; Streptococcus bovis and

colon cancer; persistent Chlamydia pneumoniae and

lung cancer; as well as Bartonella species and vascular

tumor formation [2].

The acquisition of genomic instability is a crucial

feature in tumor initiation and progression. Because

the baseline mutation rate is insufficient to account for

the multiple genetic changes required for cancer pro-

gression, tumor cells must acquire a ‘mutator pheno-

type’ that enhances the mutation frequency, and

allows the evolution from a pre-malignant to an inva-

sive cancer cell [3]. This phenotype can be caused by a

failure to repair damaged DNA and ⁄or altered activa-

tion of the DNA damage-induced checkpoint

responses that selectively eliminate irreversibly dam-

aged cells. In the case of bacterial infection, several

events (e.g. the establishment of chronic inflammation,

as well as the production of genotoxins or bacterial

products that interfere with regulation of cell cycle

Keywords

bacterial genotoxin; chronic inflammation;

colibactin, cytolethal distending toxin; DNA

damage; DNA damage response; genomic

instability; tumor induction ⁄ progression

Correspondence

T. Frisan, Department of Cell and Molecular

Biology, Karolinska Institutet, Box 285,

S-171 77, Stockholm, Sweden

Fax: +46 8 337412

Tel: +46 8 52486385

E-mail: [email protected]

(Received 1 March 2011, revised 4 April

2011, accepted 13 April 2011)

doi:10.1111/j.1742-4658.2011.08125.x

Cytolethal distending toxin, produced by several Gram-negative bacteria,

and colibactin, secreted by several commensal and extraintestinal patho-

genic Escherichia coli strains, are the first bacterial genotoxins to be

described to date. Exposure to cytolethal distending toxin and colibactin

induces DNA damage, and consequently activates the DNA damage

response, resulting in cell cycle arrest of the intoxicated cells and DNA

repair. Irreversible DNA damage will lead to cell death by apoptosis or to

senescence. It is well established that chronic exposure to DNA damaging

agents, either endogenous (reactive oxygen species) or exogenous (ionizing

radiation), may cause genomic instability as a result of the alteration of

genes coordinating the DNA damage response, thus favoring tumor initia-

tion and progression. In this review, we summarize the state of the art of

the biology of cytolethal distending toxin and colibactin, focusing on the

activation of the DNA damage response and repair pathways, and discuss

the cellular responses induced in intoxicated cells, as well as how prolonged

intoxication may lead to chronic inflammation, the accumulation of geno-

mic instability, and tumor progression in both in vitro and in vivo models.

Abbreviations

AaCDT, Aggregatobacter actinomycetemcomitans CDT; CDT, cytolethal distending toxin; DDR, DNA damage response; DSB, double strand

break; EcCDT, Escherichia coli CDT; ER, endoplasmic reticulum; H2AX, histone 2AX; HdCDT, Haemophilus ducreyi CDT; HR, homologous

recombination; MRN, Mre11-Rad50-Nbs1; NF, nuclear factor; NRPS, nonribosomal peptide megasynthetase; PARP, poly(ADP-ribose)

polymerase; PKS, polyketide megasynthetase; ROS, reactive oxygen species; StCDT, serovar Typhi CDT; Th, T helper.

FEBS Journal 278 (2011) 4577–4588 ª 2011 The Authors Journal compilation ª 2011 FEBS 4577

progression and apoptosis), in association with host

genetic factors, may contribute to the acquisition of

the mutator phenotype.

In this review, we focus on the two bacterial prod-

ucts that act as genotoxins and can directly damage

the host DNA: cytolethal distending toxin (CDT) and

colibactin. We discuss the biology of these two geno-

toxins, as well as their contribution to induction of

chronic infection ⁄ inflammation, genomic instability

and tumor progression.

CDT

CDT is the first bacterial genotoxin to be described and

is produced by a variety of Gram-negative bacteria, such

as Escherichia coli, Aggregatobacter actinomycetemcom-

itans, Haemophilus ducreyi, Shigella dysenteriae, Cam-

pylobacter sp. and Helicobacter sp, and S. enterica [4].

CDT is generally a exotoxin, and the active holotox-

in is a tripartite complex encoded by a single operon

[5,6], formed by the CdtA, CdtB and CdtC subunits

(Fig. 1A). Nesic et al. [7] solved the crystal structure of

the H. ducreyi CDT (HdCDT) and demonstrated that

the CdtA and CdtC subunits are lectin-like molecules,

sharing structural homology with the B-chain repeats

of the plant toxin ricin [7] (Fig. 1B). CDT is an

exotoxin secreted by the pathogen at the infection site.

Functional studies have identified CdtA and CdtC as

comprising essential proteins for mediating toxin bind-

ing to the membrane and internalization into target

cells [8–10].

The CdtB subunit adopts the canonical four-layered

fold of the DNase I family: a central 12-stranded b-sandwich packed between outer a-helices and loops on

each side of the sandwich [7]. The crystal structure

confirms previous data demonstrating that the CdtB

subunit is functionally homologous to the mammalian

DNase I, and also possesses DNase capacity both

in vitro and when ectopically expressed or microinjected

in eukaryotic cells. Mutation in any conserved residue

important for the catalytic activity or the Mg2+ bind-

ing abolishes the ability of CdtB to cleave DNA in vitro

and to induce DNA damage responses (DDRs) in

model cell lines [11–14].

CDT is therefore defined as an A-B2 toxin, where

CdtA and CdtC are required for binding the holotoxin

to the plasma membrane of target cells, allowing entry

of the active CdtB, which can translocate to the

nucleus and induce DNA lesions.

In addition to the well-characterized DNA damaging

activity of the CdtB subunit, Shenker et al. [15]

reported that the active subunit from A. actinomyce-

Fig. 1. Structure of CDT holotoxin and the psk genomic island. (A) Schematic representation of the CDT genes from H. ducreyi and S. enter-

ica, serovar Typhi. In all CDT-producing bacteria identified to date, excluding S. enterica, the three cdt genes are organized in an operon and

are transcribed as monocistrons. Conversely, in S. enterica, the three genes required for an active holotoxin are present as two separate

units: one unit containing the cdtB gene, encoding the active subunit, and the pltB ⁄ pltA unit, encoding proteins possibly required for the

proper traffic of CdtB to the nucleus of target cells. No homologous genes for the cdtA and cdtC subunits have been identified in this bacte-

rium. (B) Crystal structure of the HdCDT, adapted from Nesic et al. [7]. Protein data bank code: 1SR4. (C) Schematic representation of the

pks genomic island that encodes the enzymes and accessory proteins required for synthesis of an active colibactin in the E. coli strain Nissle

1917 [30].

Bacterial genotoxins and genomic instability L. Guerra et al.

4578 FEBS Journal 278 (2011) 4577–4588 ª 2011 The Authors Journal compilation ª 2011 FEBS

temcomitans exhibits PI-3,4,5-triphosphate phosphatase

activity [15]. However, there is evidence suggesting that

the DNA damaging activity alone is sufficient to con-

fer CdtB toxicity. Indeed, the G2 arrest and cell death

induced by conditional expression of CdtB in Saccha-

romyces cerevisiae depend exclusively on its DNase-

catalytic residue because yeast does not harbor the

substrate for CdtB phosphatidylinositol-3,4,5-triphos-

phate phosphatase activity [16] and specific CdtB

mutations that inhibit the phosphatase activity (but

retain DNase activity) are sufficient to induce the cell

death in proliferating U937 monocytes [17].

The discrepancy between the requirements of the dif-

ferent enzymatic activities of CDT may depend on the

cell type used as model. It is conceivable that T lym-

phocytes are more susceptible to the phosphatase

activity of CDT compared to all of the other cell lines

tested.

The only exception to the general structure of the

CDT family described so far is represented by the

S. enterica, serovar Typhi CDT (StCDT). In this intra-

cellular pathogen, the three genes required for an

active cytotoxin are not part of a single operon

because the gene for cdtB is not associated with genes

encoding for the CdtA and CdtC subunits. No homo-

logs for cdtA and cdtC genes have been found within

the complete Salmonella typhi genome [18]. However,

the toxicity of the StCDT on target cells requires the

transcription of two other genes: pltB (pertussis-like

toxin B) and pltA (pertussis-like toxin A) (Fig. 1A).

In vitro reconstitution experiments have shown that the

products of these three genes form an tripartite com-

plex inducing DNA damage in intoxicated eukaryotic

cells [19].

Interestingly, expression of the cdtB, pltB and pltA

genes is switched on upon bacterial uptake by the host

cells, and it is conceivable that the PltB and PltA sub-

units are required to transport CdtB from its site of

production within the cells to the extracellular med-

ium, from where StCDT can also intoxicate cells that

have not been infected, in a paracrine manner [19].

Several details of CDT binding to the plasma mem-

brane of target cells and its intracellular trafficking to

the nucleus have been elucidated (Fig. 2).

Interaction of the A. actinomycetemcomitans CDT

(AaCDT) occurs within GM1-enriched regions of the

plasma membrane, which are characteristic of mem-

brane rafts [14,20], and cholesterol depletion by

methyl-b-cyclodextrin reduces the ability of both

AaCDT and HdCDT to bind to Jurkat and HeLa cell

lines, respectively, and prevents intoxication [14,20].

Furthermore, inactivation of mutations within the

SGMS1 gene that reduce the levels of sphingomyelin

(a key component of lipid rafts) confers resistance to

the E. coli CDT (EcCDT) [21].

The identity of the toxin receptor is still unknown.

Fucose may represent the binding determinant for the

EcCDT-II [10], whereas another study indicated that

the AaCDT holotoxin binds to surface glycosphingoli-

pids and that inhibitors of glycosphingolipid synthesis

can prevent intoxication of the human monocytic

U937 cell line [22]. Site-directed mutagenesis of a

human cell line haploid for all chromosomes except

chromosome 8 identified mutants for the membrane-

expressed protein TMEM181 that were resistant to

EcCDT [21].

On the basis of such evidence, it is conceivable that

each individual CDT exhibits different receptor speci-

ficity. In line with this evidence, Eshraghi et al. [23]

reported that CDTs from H. ducreyi, A. actinomyce-

temcomitans, E. coli and Campylobacter jejuni differ in

their abilities to intoxicate host cells. The binding of

Aa, Hd and EcCDT-III, but not CjCDT, is dependent

on the presence of cholesterol. Unexpectedly, mutant

Chinese hamster ovary cells that lack N-linked com-

plex and hybrid carbohydrates, as well as cells that

lack glycosphingolipids or are deficient in fucose bio-

synthesis, are as similarly sensitive as the wild-type to

intoxication by all four CDTs tested, indicating that

N- and O-glycan, or fucosylated structures are dispens-

able for mediating toxin binding [23].

Upon binding to the plasma membrane, the HdCDT

is internalized in HeLa cells by dynamin-dependent

endocytosis, and it further transits to the endosomal

compartment [24]. Biochemical and imaging experi-

ments have demonstrated that the toxin is then retro-

gradely transported via the Golgi complex to the

endoplasmic reticulum (ER) [14]. Other bacterial and

plant toxins have been described to traffic from the

plasma membrane to the ER (Shiga, cholera and

ricin), and they subsequently reach their targets in the

cytosol by retrograde transport via the ER degradation

pathway (Fig. 2). However, HdCDT could not be

detected by biochemical assays in the cytosol of intoxi-

cated cells [25]: this opens the possibility that the CDT

active subunit may translocate directly from the ER to

the nucleus. Two studies have identified specific

nuclear localization signals (NLS) within AaCdtB and

EcCdtB-II. In AaCdtB, a nonconventional NLS is

localized at the N-terminus of the protein, and the

deletion of 11 amino acids within the this sequence

abolishes intoxication [26]. Conversely, two NLS

sequences, designated as NLS1 and NLS2, have been

identified in the carboxy-terminal region of the

EcCdtB-II [8]. Interestingly, the deletion of each of

these sequences produces a differential localization of

L. Guerra et al. Bacterial genotoxins and genomic instability


the active toxin subunit, suggesting that they play dif-

ferent roles in the intracellular trafficking of EcCDT-

II. Cells intoxicated with a holotoxin containing the

EcCdtB-II-DNLS1 display a perinuclear distribution,

which is consistent with trapping of the active toxin

component in the late endosome and ⁄or ER com-

partment, whereas a diffuse cytoplasmic staining is

observed in cells exposed to the EcCdtB-II-DNLS2-

containing toxin. It is conceivable that the NLS1

promotes the ER-to-nucleus translocation, whereas

NLS2 may function as an ER compartimentalization

signal preventing the escape of the CdtB molecules to

the cytosol, allowing their transit to the nucleus. It is

still not known why the presence of the putative NLS

domains are so divergent in AaCDT and EcCDT-II

because the structure of CdtB subunits presents a high

degree of conservation among different bacteria [27].

On the basis of such evidence, it is clear that the

nuclear translocation of the CdtB subunit into the

nucleus remains an open issue. Another interesting

question is whether the CdtA and CdtC subunits assist

the active component in its trafficking within the host

cells.

Colibactin

This toxin has been recently characterized and very

little information is available regarding its biology.

Colibactin is a putative hybrid peptide–polyketide

genotoxin found in both commensal and pathogenic

bacteria. Colibactin has been mostly characterized in

extraintestinal pathogenic E. coli strains of the phylo-

genetic group B2 [28]. The enzymes required for the

synthesis of colibactin are present in a 54 kb genomic

island, referred to as the pks island, located in the

asnW tRNA locus. This region contains 23 putative

ORFs, including three nonribosomal peptide megasyn-

thetases (NRPS), three polyketide megasynthetases

Fig. 2. Summary of the CDT internalization pathway and cellular responses induced by bacterial genotoxins. Binding of CDT is dependent on

the presence of intact lipid rafts, and the toxin is internalized via dynamin-dependent endocytosis into early and late endosomes. At this

stage, it is not known whether the CdtA (violet) and ⁄ or the CdtC (pink) subunits are internalized and follow the active CdtB subunit (green)

into the nucleus. The CdtB subunit further transits to the Golgi complex, and is then retrogradely transported to the ER. The mechanisms of

nuclear translocation have not yet been fully elucidated. Once in the nucleus, the CdtB subunit causes DNA damage and activates the ATM-

dependent DNA damage response, characterized by recruitment of the MRN complex, and full activation of ATM at the site of the damage,

which also requires a functional c-MYC. Activation of ATM promotes phosphorylation of histone H2AX and activation of the DNA damage

checkpoint responses via activation of: (a) the tumor suppressor p53 and its downstream effector p21, which results in G1 arrest, and (b)

the kinase CHK2 that blocks cell proliferation in the G2 phase of the cell cycle by inactivating the CDC25 phosphatase, resulting in hyper-

phosphorylation and inactivation of the cyclin-dependent kinase CDK1 (pCDK1). CDT intoxication also activates RhoA-dependent survival sig-

nals. This effect requires a functional ATM and is dependent on dephosphorylation of the guanine nucleotide exchange factor Net1.

Activation of RhoA regulates two distinct pathways: (a) induction of actin stress fibers, which requires the RhoA kinases ROCKI and ROCKII,

and (b) activation of p38 mitogen-activated protein kinase, associated with delayed cell death.



(PKS), two hybrid NRPS ⁄PKS megasynthetases, and

ten accessory, tailoring and editing enzymes (Fig. 1C).

Mutation analysis demonstrated that all the PKS and

NRPS and eight of the accessory genes are required

for production of an active genotoxin [28].

Screening studies have demonstrated that the pks

island is also present in other members of the Entero-

bacteriaceae family, such as Klebsiella pneumoniae,

Enterobacter aerogenes and Citrobacter koseri isolates

[29].

Gene expression of the ORFs required for the colib-

actin synthesis has been studied, using the nonpatho-

genic E. coli strain Nissle 1917 as a model, by

Homburg et al. [30], who identified seven transcripts,

four of which are polycistrons. The polycistronic tran-

scripts comprise the genes: (a) clbC to clbG; (b) clbI to

clbN; (c) clbO to clbP; and (d) clbR to clbA, whereas

the other ORFs are transcribed as monocistrons [30].

Luciferase reporter assays performed on the clbA,

clbB, clbQ and clbR genes demonstrated that their

expression increased during late logarithmic and early

stationary phase of the bacteria growth course. The

levels and the duration of expression depend on the

culture medium used, with DMEM being the best con-

dition compared to LB or minimal medium supple-

mented with 0.2% glucose [30]. Interestingly, the

transcription of these ORFs was not induced by direct

contact with the mammalian cell line HeLa [30]. It

remains to be determined whether the expression of

the other clb genes is dependent on interaction with

eukaryotic cells.

DDRs and genomic instability

CDT possesses DNase activity in vitro, and both CDT

and colibactin cause DNA damage in intoxicated cells

[11–14,28]. Thus, before discussing the cellular

responses to these genotoxins, we briefly review the

state of the art of the DNA damage sensing and repair

pathways in mammalian cells, as well as the conse-

quences of an altered DDR in the promotion of geno-

mic instability. On the basis of the type of DNA

damage induced by CDT and colibactin, we focus

mainly on the cellular responses to DNA double

strand breaks (DSB).

DDRs are essential for preserving the genetic infor-

mation and maintain genomic integrity in cells exposed

to the damaging activity of endogenous [e.g. reactive

oxygen species (ROS) produced by the cellular metab-

olism] and environmental agents (ionizing radiation

and UV radiation). Sensing and activation of the

DDR is coordinated by proteins of the phosphatidyl-

inositol 3-kinase-like protein kinase family: ATM,

ATR and DNA-PK [31]. The outcome is a block of

cell cycle progression and activation of the DNA dam-

age repair pathways. Successful repair will allow the

cell to resume the normal cell cycle progression,

whereas damage beyond the possibility of repair will

promote either apoptosis or senescence, precluding the

survival and replication of cells that can accumulate

genomic instability [32]. A key effector protein that

regulates activation of cell death or senescence in the

presence of chronic and unrepaired DNA damage is

the tumor suppressor gene p53, which acts as a barrier

for cancer initiation ⁄progression [33].

Many DNA repair pathways have been evolved to

cope with all the possible insults to which the cellular

DNA is exposed. A mismatch DNA base is replaced

with the correct one by the mismatch repair [34],

whereas small base alterations, such as alkylation, are

repaired by the base excision repair, which removes

the altered base [35]. More complex lesions, such the

UV-induced pyrimidine dimers, require a longer exci-

sion (approximately 30 bp) and are repaired by the

nucleotide excision repair pathway [36]. DSB will be

processed by nonhomologous end joining or homolo-

gous recombination (HR) [37]. Nonhomologous end

joining occurs throughout the cell cycle and is based

on the identification of the break and subsequent

rejoining of the two ends. Consequently, there is a loss

or addition of nucleotides at the site of the lesion, and

the repair mechanism per se can contribute to a certain

degree of genomic alteration. Conversely, HR per-

forms an error-free repair of the lesion because the sis-

ter chromatid is used as template, restricting this type

of repair mechanism to the late S and G2 phases of

the cell cycle.

Recognition of DSB is mediated by several sensor

complexes: members of the poly(ADP-ribose) polymer-

ase (PARP) family, specifically PARP1 and PARP2,

the Mre11-Rad50-Nbs1 (MRN) complex and the

Ku70 ⁄Ku80 hetorodimer [31].

PARP1 and 2 are activated by DNADSB and catalyze

the addition of poly(ADP-ribose) chains on histones

and nuclear proteins. This step is essential for the

recruitment of the MRN complex, which initiates a

resection of the DNA ends to produce a 3¢ tail, and

promotes the accumulation and full activation of the

ATM kinase at the site of the damage [38]. In turn,

ATM coordinates the full DNA resection to promote

HR, and activates the checkpoint responses to block

cell cycle progression, allowing repair and preservation

of the genomic integrity. This is achieved by: (a)

recruitment and phosphorylation of c-histone 2AX

(H2AX) important to sustain the DDR; (b) activation

of effectors such as CtlP, BRCA1, ARTEMIS



(DNA resection); and (c) phosphorylation ⁄ activationof CHK2 and p53 that lead to cell cycle arrest and, in

ultimate instances, to apoptosis or senescence

[31,32,39].

Upon extensive DNA resection, the RPA complex

binds to the 3¢ ssDNA ends and modulates the activity

of effectors, such as BRCA2 and Rad51, which execute

the recombination process by performing strand inva-

sion and Holliday junction formation and resolution

to produce two completed undamaged sister chromat-

ids that preserve the original genetic information [31].

Figure 3 summarizes the main events in the ATM-

dependent activation of the DDR.

In nonhomologous end joining, the end of the

breaks are recognized and bound by the Ku79 ⁄Ku80

heterodimer, which in turn recruits the DNA-PK and

initiates the DNA resection process. After binding to

the DNA, the DNA-PK is autophosphorylated and

provides access to the site of the resecting protein

ARTEMIS, and also the ligase complex XRCC4 ⁄Lig4,which promotes re-ligation of the broken ends [40].

Genomic instability is a characteristic of almost all

human tumors. The major genomic alterations

described in cancer cells include chromosomal instabil-

ity and microsatellite instability [41]. Chromosomal

instability is characterized by losses of entire or large

portions of chromosomes, resulting in aneuploidy,

translocation and loss of heterozygosity, whereas

microsatellite instability is defined as an expansion or

contraction of the number of oligonucleotide repeats

present in microsatellite sequences apart from the

nucleus.

The importance of the DDR in the maintenance of

genomic integrity is highlighted by the demonstration

that the acquisition of genomic instability is linked to

mutations in genes controlling DNA repair and mitotic

checkpoint pathways in hereditary cancers [42].

Conversely, high throughput analysis demonstrated

that, in sporadic cancers, the most frequently altered

genes are the TP53 tumor suppressor and genes that

regulate cell growth either positively (e.g. the oncoge-

nes EGFR and RAS) or negatively (e.g. PTEN and

CDKN2A) [42]. This pattern is not unexpeceted

because deregulation ⁄overexpression of oncogenes will

lead to DNA replication stress and stalled replication,

resulting in the formation of DNA damage and activa-

tion of DDR. One of the consequences of this will be

the activation of p53 and the promotion of cell death

or senescence, a response that will pose a barrier to

tumor initiation ⁄progression. Therefore, only cells in

which the deregulation of oncogenes is accompanied

by alteration of the p53 tumor suppression pathway

will have the possibility of overcoming this barrier.

Cellular responses to colibactin andCDT

We now discuss the key cellular responses activated in

cells exposed to CDT or colibactin-producing bacteria,

focusing on the DDR and cell survival, which are both

relevant in the context of maintaining genome integ-

rity. For a more detailed analysis of the CDT-induced

cellular responses, several comprehensive reviews on

CDT biology are available [4,43,44].

Fig. 3. Summary of the ATM-dependent

DNA damage responses to DNA DSBs.

Induction of DNA DSBs activates PARP1,

which mediates the initial recruitment of the

MRN complex and promotes the full activa-

tion of ATM. In turn, ATM coordinates the

DNA damage repair resulting in: (1) sus-

tained DDR by phosphorylating histone

H2AX; (2) resection of the damaged DNA to

allow activation of the HR process; and (3)

activation of the checkpoint responses that

block cell proliferation in distinct phases of

the cell cycle (G1 or G2) to allow repair. If

the damage is beyond repair, this response

will result in elimination of the altered cell

by apoptosis or the induction of cellular

senescence, a p53-dependent process

defined as the tumorigenesis barrier.



It is now well established that CDT acts as a nucle-

ase cleaving DNA substrates in vitro, inducing nuclear

fragmentation and chromatin disruption when trans-

fected in cultured mammalian cells or Saccharomy-

ces cereviase, and promoting DNA fragmentation in

cells exposed to purified soluble toxin [11–13,45]. Simi-

larly exposure of mammalian cells to E. coli strains

expressing the pks island promotes DNA fragmenta-

tion, as detected by the comet assay [28].

As a result of the DNA damaging activity, intoxi-

cated cells activate the classical DDR, which includes

recruitment of the DNA damage sensor complex

MRN and the ATM kinase, phosphorylation of his-

tone H2AX, activation of p53 and its transcriptional

target, the cyclin-dependent kinase inhibitor p21, and

phosphorylation of checkpoint kinase CHK2. Tran-

scriptional upregulation of p21 leads G1 arrest,

whereas CHK2-dependent inactivation of the CDC25

phosphatase leads to an accumulation of the hyper-

phosphorylated form of cyclin-dependent kinase 1

(CDK1, also known as CDC2), and consequent induc-

tion of G2 arrest [9,46–51].

The prompt activation of the ATM-dependent

response to CDT or ionizing radiation-induced DNA

damage also requires a functional proto-oncogene

MYC [52].

As a consequence of the activation of ATM-depen-

dent checkpoint responses, cells exposed to CDT or

colibactin stop proliferating [28,46,51]. Furthermore,

CDT-intoxicated normal or tumor cells express the

hallmarks of cellular senescence, such as persistently-

activated DNA damage signaling (detected as

53BP1 ⁄cH2AX-positive foci), enhanced senescence-

associated b-galactosidase activity, and expansion of

promyelocytic nuclear compartments [53]. In support

of the DNA damaging activity of CDT, a genome

wide analysis performed in S. cerevisae identified HR

and activation of the DNA damage checkpoints as

comprising essential mechanisms for the response to

damage induced by the conditional expression of the

active CdtB subunit from C. jejuni [54].

Another important aspect in the context of bacterial

genotoxins and carcinogenesis is the activation of sur-

vival signaling pathways because the survival of cells

with damaged DNA enhances the risk of acquiring

genomic instability and favors tumor initiation and ⁄orprogression [55,56] (Fig. 4). The survival of CDT

intoxicated cells is dependent on the activation of the

small GTPase RhoA [45], which induces actin stress

fiber formation via the RhoA kinases, ROCKI and

ROCKII, and prevents cell death via activation of the

mitogen-activated protein kinase p38 and its down-

stream target mitogen-activated protein kinase-acti-

vated protein kinase 2 [57] (Fig. 2). The activation of

RhoA is dependent on the dephosphorylation on ser-

ine152 of the RhoA-specific guanine nucleotide

exchange factor Net1, and it appears to be part of the

DDR response because it requires a functional ATM

[57].

The cellular responses to the two bacterial toxins are

summarized in Fig. 2.

Infection with CDT-producing bacteria:chronic inflammation and tumorprogression

Over the past 10 years, chronic inflammation has been

shown to be associated with an enhanced risk of tumor

development. How can inflammation favor the acquisi-

tion of the mutator phenotype? The inflammatory

environment is characterized by the production of

ROS and reactive nitrogen intermediates. These com-

pounds are potent genotoxic agents that may increase

the mutation rate and promote the accumulation of

genomic instability, thus altering the crucial biological

processes such as the regulation of DNA repair and

DDRs, allowing tumor initiation ⁄progression [58,59].

Chronic inflammation is also associated with constit-

utive activation of pleiotropic nuclear factor (NF)-jB,

Fig. 4. Possible role of bacterial genotoxins in cancer development.

Chronic infection with CDT or colibactin-producing bacteria can pro-

mote the induction of genomic instability by direct secretion of bac-

terial genotoxins and activation of a chronic inflammation, which is

associated with the production of endogenous DNA damaging

agents, such as ROS. Persistent activation of the transcription fac-

tor NF-jB, via the pro-inflammatory cytokine TNF-a or sustained

triggering of pathogen recognition receptors (e.g. Toll-like recep-

tors), in combination with survival signals induced by cellular intoxi-

cation with genotoxins, may allow cells carrying genomic instability

to break through the tumorigenesis barrier, resulting in an increased

risk of tumor development.



which promotes cell survival, and has been demon-

strated to contribute to tumor formation in models of

colitis-associated cancer and hepatocellular carcinoma

[60,61].

Bacteria that cause persistent infections associated

with chronic inflammation have a higher risk of pro-

moting carcinogenesis [58]. The best-studied example is

H. pylori and its association with gastric carcinoma

and mucosa-associated lymphoid tissue lymphoma

[62]. Chronic infection with this bacterium is associ-

ated with the downregulation of mismatch repair and

base excision repair proteins both in vitro and in vivo

[63], and these effects correlate with a five-fold

increased mutation frequency and also the induction of

microsatellite instabilities in the gastric epithelium of

mice 6 months after infection [64]. Epidemiological evi-

dence also demonstrate an increased risk of carcinoma

of the gallbladder in chronic carriers of S. enterica, ser-

ovar Typhi [2] and colon cancer in individuals colo-

nized by Bacteroides fragilis [65]. Bacterial products

can contribute to a sustained and deregulated inflam-

matory microenvironment by stimulation of the host

pathogen recognition receptors, leading to a constant

supply of ROS, reactive nitrogen intermediates and

cytokines. The most well-characterized family of PPRs

is the Toll-like receptor family. The majority of Toll-

like receptor signaling converges to the adaptor

molecule MyD88 and activates the transcription factor

NF-jB. MyD88 knockdown was shown to strongly

reduce the development of spontaneous colorectal

carcinoma in mice carrying heterozygous mutation of

the tumor suppressor genes APC (ApcMin ⁄ + mouse

model) [66]. In addition to the contribution of the

innate immune responses in inflammation-induced car-

cinogenesis via stimulation of PPR or pro-inflamma-

tory cytokine production, there is now evidence linking

T cell-mediated immune responses in infection-induced

carcinogenesis. The production and secretion of the

B. fragilis toxin induces colitis, which further develops

into colon cancer in the ApcMin ⁄ + mouse model, and

the carcinogenic capacity of B. fragilis toxin-producing

strains is directly associated with the recruitment of

the highly pro-inflammatory subset of T helper (Th) 17

lymphocytes [67].

Figure 4 summarizes the effectors that may contrib-

ute to carcinogenesis in chronic bacterial infections.

Considering the importance of establishing a chronic

infection as risk for tumor development, several studies

have assessed whether CDT may contribute to persis-

tent bacterial colonization of the gastrointestinal tract.

Fox et al. [68] demonstrated that a functional toxin

favors colonization of the stomach and lower bowel

of C57BL ⁄ 129 mice infected with C. jejuni. The

C57BL ⁄ 129 mouse model was not suitable for studying

the effect of a persistent infection with CDT-producing

bacteria in the induction of inflammation because bac-

teria were detected only in a proportion of the mice.

Conversely, a persistent colonization of the stomach

and the lower bowel for 100% of animals was achieved

in C57BL ⁄ 129 mice carrying a homozygous deletion of

p50 and heterozygous deletion of the p65 subunits of

the transcription factor NF-jB (p50) ⁄ ) p65+ ⁄ )). In

this model of chronic infection, the presence of CDT-

producing bacteria was associated with significantly

enhanced severity of the gastritis and a greater induc-

tion of gastric hyperplasia and dysplasia, which is an

indication of an early neoplastic process [68].

Colonization of the Swiss Webster mice with Heli-

cobacter hepaticus was also dependent on expression of

a wild-type CDT [69]. This persistent infection was

associated with a highly inflammatory response, char-

acterized by the production of Th1-associated IgG2a,

Th2-associated IgG1 and mucosal IgA [69].

Similarly, a strong inflammatory response was

described in the liver of male A ⁄ JCr mice infected with

H. hepaticus carrying a wild-type CDT 10 months after

infection compared to mice infected with an isogenic

strain carrying a mutant toxin, where the cdtB gene

was inactivated by transposon mutagenesis. This

response was characterized by an increase in transcrip-

tion levels of pro-inflammatory (TNF-a, IFN-c and

Cox-2, IL-6 and TGF-a) and anti-apoptotic (Bcl-2 and

Bcl-XL) genes, as well as upregulation of hepatic

mRNA levels of components of the NF-jB pathway

(p65 and p50) [70]. The presence of CDT was further

associated with a progression of inflammation to dys-

plasia. The dysplastic lesions were characterized by the

presence of hepatocytes with a marked variation in cell

and nuclear size and shape, as well as a loss of hepatic

architecture [70].

An interesting notion, providing fuel for future stud-

ies, is to assess whether these conditions of dysplasia

are associated with the CDT-dependent induction of

DNA damage, chronic activation of the DDRs, acqui-

sition of genomic instability and alteration of cellular

pathways that regulate senescence, allowing cells to

break through the tumorigenesis barrier. Indeed, very

little is known about the ability of CDT to induce

genomic instability.

Effects of colibactin on genomicinstability

The effects of colibactin in induction of DNA damage

in vivo and genomic instability in vitro were studied by

Cuevas-Ramos et al. [50]. These authors reported that



the expression of the clbA gene, a key gene for the syn-

thesis of this genotoxin, was detected in a mouse intes-

tinal loop model and in the colons of antibiotic-treated

BALB ⁄ cJ mice 6 h and 5 days, respectively, after infec-

tion with an E. coli strain harboring the psk genetic

island. In each case, an isogenic strain carrying a clbA

mutant gene (and therefore unable to produce colibac-

tin) was used as negative control. The expression of

the clbA gene was further associated with the induction

of DNA damage, as assessed by increased phosphory-

lation of the histone H2AX [50].

Short-term exposure of the Chinese hamster ovary

cell line to psk+ E. coli at low multiplicity of infection

(in the range five to 20 bacteria per cell) induced DNA

damage that could still be observed, although at a very

low level, up to 24 h post-infection in actively cycling

cells, indicating that the DNA repair process was not

completed. As a consequence of the partial DNA

repair, the infected cells presented anaphase bridges,

accumulated chromosomal aberrations in approxi-

mately 7% of the chromosomes, with ring chromo-

somes and translocations being the most common

alterations, and aneuploidy (a loss or gain of chromo-

somes) 72 h post-infection. Such aberrations were

maintained in a proportion of cells up to 21 days post-

infection. The chromosomal instability induced by

infection with colibactin-producing E. coli was further

associated with an enhanced rate of mutation fre-

quency and an increased ability of the cells to grow in

soft agar, which is a feature of anchorage-independent

growth [50].

Future perspectives

Our knowledge on how bacterial infections can con-

tribute to carcinogenesis has begun to be unraveled.

The journey started from epidemiological data and

several molecular mechanisms have been identified to

date, with special focus on the oncogenic role of

H. pylori infection. The identification of bacterial

genotoxins opens yet another new avenue.

We have come a long way in our understanding of

the biology of CDT, although many questions still

remain. We do not know: (a) when and under what

conditions the toxin is produced in vivo; (b) what is the

extent of the DNA damage and genomic instability in

in vivo models; and (c) whether there is a correlation

between chronic infection of CDT-producing bacteria

and an increased risk of cancer development. A retro-

spective study demonstrated that infection with the

enteropathogenic C. jejuni, where 99% of the strains

harbour cdt genes, did not correlate with an increased

risk of developing a tumor in the gastrointestinal tract

at least during the first 10 years after the detection of

infection [71]. However, this bacterium is rarely associ-

ated with the establishment of a chronic infection and

therefore may not represent a suitable model, despite

the fact that C. jejuni was found in tissue specimens

derived from intestinal mucosa-associated lymphoid

tissue lymphoma patients [72].

The biology of colibactin is still at its infancy

because this toxin was only characterized recently

and cannot yet be produced as a synthetic molecule

in vitro. Several interesting questions remain: (a) how

does it induce DNA damage; (b) how does it enter and

traffic within the host cells to reach the nuclear

compartment; and (c) does it contribute to long-term

bacterial colonization in vivo.

As a more general evolutionary aspect, we still do

not know how bacteria benefit from producing such

genotoxins.

The experimental work in the field of bacteria and

cancer, and specifically on bacterial genotoxins, has

been hampered by the complexity of the host–bacteria

interaction, although the development of suitable ani-

mal models and the implementation of high through-

put screenings will provide conditions that allow the

pursuit of this exciting issue in the field of medical

science.

Acknowledgements

This work was supported by the Swedish Research

Council, the Swedish Cancer Society, the Ake-Wiberg

Foundation, the Magnus Bergvall Foundation, the

Karolinska Institutet to T.F., and the Robert Lund-

berg Memorial Foundation to L.G. T.F. is supported

by the Swedish Cancer Society.

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