E. coli and colorectal cancer: a complex relationship that deserves a critical mindset

Figures & data

Table 1. Cyclomodulins of E. coli.

Cyclomodulins	Gene(s)	Mechanism	Reference
Cytolethal Distending Toxin (CDT)	cdtA, cdtB, and cdtC	DNA double-strand breaks caused by CdtB arrest the cell cycle	Lara-Tejero and Galan (2000)
Intimin-dependent attachment	eae, Type III secretion system	Effector eae down-regulates the DNA mismatch repair system, resulting in DNA strand breaks	Maddocks et al. (2009)
Cell cycle inhibiting factor	cif		Marchès et al. (2003)
Cytotoxic necrotizing factor	cnf1	Deamination of Rho-GTPase results in actin cytoskeleton activation, resulting in multinucleation	Fabbri et al. (2013)
Colibactin	pks locus	Introduction of DNA strand breaks due to reactive cyclopropane group	Nougayrède et al. (2006)

Figure 1. Schematic representation of the pks locus of E. coli. Genes coding for polyketide synthases are shown in dark grey, non-ribosomal peptide synthase genes are shown in light grey, and the two fusion genes are shown bicoloured. The regulator clbR and the integrase (int) are shown in black. Protein functions for ClbA, ClbM, and ClbP are indicated. Modified after Nougayrède et al. (2006) and Homburg et al. (2007). Small accessory genes are not shown.

Table 2. Mouse models demonstrating cancer-inducing properties of E. coli.

Model	Colonization	Comparison	Result	Reference
Germfree IL^−10 mice treated with carcinogen	Monocolonization results in tumours	Mouse strain NC101 vs. isogenic pks^− mutant	More rapid and larger tumours in treatment group than in control	Arthur et al. (2012)
C57B/6J-Apc^−/+ mice (Min mice) treated with streptomycin	Colonization results in tumours	Human CRC strain 11G5 vs. K-12 strain MG1665	More colonic polyps in treatment group than in control	Bonnet et al. (2014)
Colon-loop colonization	Six-hour presence in closed loops results in DNA strand breaks	human strain SP15 vs. isogenic pks^– mutant	3x more DNA strand breaks in colonocytes of treatment than in control	Cuevas-Ramos et al. (2010)
Subcutaneous injection of tumour cells	Tumour cells were infected with E. coli containing pks on pBAC	E. coli strain DH10B with pBAC containing pks from strain CCR20 vs. empty pBAC	Larger tumours in treatment than in control due to growth factors	Cougnoux et al. (2014)
Xenografts of HC116 cells	Xenograft cells were infected with E. coli	Unspecified E. coli strain with or without pks	Tumors are suppressed by high MOI, stimulated by low MOI, only with pks^+ strain, via cytokines	Dalmasso et al. (2014)

Figure 2. Proposed structures of colibactin and pre-colibactin. (A) The originally proposed structure of pre-colibactin (top), colibactin A after cleavage by ClbP (middle) and colibactin cross-linked with two sites to DNA (bottom), after (Vizcaino and Crawford 2015). (B) The revised structure of pre-colibactin A, and derivatives pre-colibactin B and C (insets) after (Healy et al. 2016). The part abbreviated with “R” remained unrevised and is thus identical to what is shown to the left. (C) The revised structure of colibactin A, with an imine group (the 5-ring to the left of the molecule) that presumably enhances DNA reactivity, after (Healy et al. 2016). (D) The structure of colibactin according to (Li et al. 2016).

Table 3. Observations pro and against the carcinogenic effect of E. coli colibactin.

Observation	For	Against
Epidemiological observations	Both CRC and carriage of E. coli phylotype B have recently increased	Correlation does not necessarily equal causation
Human biopt investigations	CRC lesions are frequently heavily colonized by E. coli, including phylotype B2 types and pks-positive strains	Colonization can be a result instead of a cause of lesions. Colonization increases as tumour development progresses
Mouse models	Increased carcinogenic effects were observed with pks-positive strains in various mouse models	All models are highly artificial. Tumour growth is suppressed at high MOI. Host-derived factors from exposed cells have similar effects
In vitro assays	pks-positive strains crosslinks purified DNA and induces DNA strand breaks in tissue culture cells	DNA crosslinking is not observed in vivo. Exposed cells produce unexplained factors enhancing the toxic effect
Mechanistic predictions	The predicted structure of colibactin is highly reactive against DNA	The predicted toxin would be equally reactive to other biomolecules. Its pre-toxic form would be as active as the spliced product

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