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Bacteria, Biofilms, & DNA Damage

Pathogenic bacteria residing in our gut produce tumorigenic genotoxins

The mammalian gastrointestinal (GI) tract is the largest immune organ in the body and hosts 70-80% of immune cells [14]. The GI tract has 3 layers of defense against pathogens: the mucosal lining, the epithelium and the residing microflora [15]. This microflora comprises about 100 trillion bacteria, both good and bad, and the bad ones have been increasingly linked to colorectal cancer, more specifically through their genotoxic products [16, 17].

Pathogenic bacterial aggregates enclosed in a matrix of self-produced extracellular polymeric substances (i.e., saccharides) are commonly referred to as biofilms, and have been frequently reported in patients with CRC. Biofilms are inflammatory structures that adhere to the mucosal layer of the GI tract and possess shielding properties that serve the harbored microbes by protecting them from altered pH, nutrient scarcity, shear forces and antibiotic accessibility [18-21]. Studies showed bacterial biofilms to be present in 50% of CRC compared to less than 15% in healthy patients [22, 23]. Close to 90% of proximal (right-sided) and over 10% of distal (left-sided) CRC samples in this breakthrough study demonstrated a significant presence of biofilms in their GI lining, the composition of which was Bacteroidetes and Fusobacterium-heavy [22, 24]. Bacteroidetes and Fusobacterium are both natural gut inhabitants, with bacteria in the genus Bacteroidetes accounting for around 50% of the resident microbiota. Bacteroidetes colonize the entire GI tract and are fierce competitors equipped with high tolerance for stresses such as gastric acid and digestive enzymes, so their overgrowth represents a health concern [25].

Findings highlighting the prevalence of biofilms rich in co-localized Bacteroides fragilis (B. fragilis) and polyketide synthetase-positive (pks+) Escherichia coli (E. coli) in patients with Familial Adenomatous Polyposis (FAP), a precancerous syndrome with almost a 100% risk of CRC development if left untreated [26], have strongly implicated these pathogen-heavy biofilms in colorectal tumorigenesis. In pre-clinical mouse models, the co-expression of pks+, i.e. toxin-secreting, E. coli and enterotoxigenic B. fragilis showed a synergistic mode of action by the two pathogens resulting in promoted tumor onset in immune-compromised mice [27].

Clostridioides difficile (C. difficile) is a pathogenic bacterial strain known to cause diarrhea and colon inflammation. According to the Centers for Disease Control and Prevention (CDC), C. difficile affects an estimated 500,000 people in the U.S. every year – infections that largely prove difficult to clear – which is twice the number of annual E. Coli illnesses. Recent findings implicate C. difficile in driving the onset of CRC, which might be one of the contributing factors underlying the alarming surge in CRC cases in patients under the age of 50 in recent years [28].

Pathogenic bacteria have been shown to release genotoxins implicated in driving oncogenic mutations [16]. Colibactin is a DNA damage-inducing toxin secreted by pks+ E. coli strains. This damage, mostly in the form of DNA inter-strand cross-links, triggers genomic instability and promotes DNA mutations in affected cells [29, 30]. Two characteristic mutational subsets, in the form of insertion-deletions (indels) and single base substitutions, that directly arise from colibactin exposure have been found to perfectly match ones found in around 4.4% and 5% of CRC cases, respectively [31]. More than 5% of the inactivating mutations in APC, a critical tumor suppressor and the most mutated gene in CRC, matched the same colibactin mutations [31].

In similar fashion to colibactin, the cytolethal distending toxin (CDT) produced by Campylobacter jejuni (C. jejuni) causes DNA damage. The mechanism for this toxin, however, is different; it instigates chromosomal instability through its deoxyribonuclease (DNase) activity [32]. Another toxin with oncogenic properties is B. fragilis toxin (BFT), the product of enterotoxigenic B. fragilis (ETBF). This toxin has proteinase rather than DNase activity, so it cleaves proteins instead of DNA, and one of its targets is the tumor suppressor E-cadherin. By degrading this anti-tumorigenic protein in neighboring GI epithelial cells, BFT drives uncontrolled cell proliferation – a hallmark of cancer [1, 33, 34].