How a bacteria from your mouth can promote cancer in your colon!

Cancer is a vastly complicated phenomenon. Cancerous tumours can arise from any tissue of the body and the causes of tumorigenesis are even more variable than the tumour types. Innumerable environmental factors have been linked to cancer; some evidently more relevant than others[1]. Tumour cells can be actively destroyed by the body through a type of white blood cell called natural killer (NK) cells [2] . Like all cells these have an array of cell surface receptors through which they can communicate with other cells and detect stimuli within the extracellular environment. All receptors require a particular molecule, or a range of select molecules, to adhere to their binding site for activation. These are referred to as ligands[3]. A fundamental innovation of the immune system is the capacity to produce a vast range of cell surface receptors. NK cells possess two particular subsets of receptors. Depending on which subset is predominantly activated, the NK cell will be triggered into killing target cells (becoming cytotoxic) or remain deactivated. These receptor subsets are aptly referred to as activator or inhibitory receptors[2]. NK cells are not explicitly used for killing tumour cells. Bacteria, parasites and viruses can all be destroyed by the cytotoxic activity of NK cells [2]. Any disruption to the signals received by NK cells can result in a suboptimal immune system, with harmful cytotoxic targets remaining intact.

A recent study by Gur et al uncovered a remarkable connection between a common oral dwelling bacterium, our immune system, and tumour survival. The bacterium in question, Fusobacterium nucleatum, is traditionally known for its contribution to the development of gum disease[4]. However over the last decade this seemingly harmless microbe has been found to play a key role in preterm births and to frequently inhabit colon cancers [5,6].  With additional knowledge that F.nucleatum can interfere with our immune system Gur, and his colleagues at the University of Jerusalem, decided to investigate this bacterium further [7,8]. They wanted to know whether this species of bacteria could provide a survival advantage to the tumours it so commonly resides in.

Gur et al began by showing that F.nucleatum could efficiently colonise several tumours of different origin; corroborating the previous literature. Next they turned their heads to assessing the effects of F.nucleatum on NK cell cytotoxicity. Various tumour types, including F.nucleatum’s seemingly favoured colon cancer, were incubated with activated NK cells for 5 hours. After incubation the percentage killing was calculated by comparing the starting number of tumour cells with the number of survivors post NK cell incubation. This was repeated with the same tumour types but this time the tumours were prexposed to F.nucleatum. In all of the prexposed tumours there was a striking decrease in NK cell killing compared to the non-exposed tumours of the same tumour type[4]. To ensure this was not a strain specific phenomenon they repeated the same experiment with another strain of F.nucleatum. Again the prexposed tumours exhibited significantly lower numbers of cells being killed[4]. To check the killing inhibition was not simply the consequence of bacterial residence, they repeated this experiment with a completely different species. In this final test with E.coli, there was no difference in the percentage killing between non-exposed and pre-exposed tumours[4]. The data strongly suggested that the bacterial species of F.nucleatum interferes with the potency of NK cytotoxicity. But how does it do it?

It was hypothesised that F.nucleatum activates an inhibitory receptor common to all NK cells. This would result in cytotoxic repression and could explain the observed survival advantage of F.nucleatum invaded tumours. To date only one inhibitory receptor is known to be expressed by all NK cells. This receptor is called TIGIT and it became the main focus of their investigation [9]. The TIGIT receptor spans the cell membrane from its external binding site, to its cytoplasmic tail. Once activated by a ligand the cytoplasmic tail conveys the external message to intracellular effectors. These quickly instigate a cease to the cell’s cytotoxic activity. A cleverly designed assay allowed Gur et al to show that F.nucleatum can activate TIGIT receptors[4]. Using genetic manipulation they fused the extracellular component of TIGIT to the cytoplasmic tail of a mouse immune receptor called CD3. This chimeric protein was then actively expressed in a cell line. The swapping of cytoplasmic tails was necessary because the CD3 tail gives a clearer, more easily measured response to receptor activation. A protein called IL-2 is released when the CD3 tail is activated. It is this protein that was used to quantify TIGIT activation[10,4]. When F.nucleatum was finally exposed to cells expressing the chimeric TIGIT receptor, IL-2 was detected[4]. This provided strong evidence that F.nucleatum can both interact and activate TIGIT.

Further experimentation was required to confirm that the F.nucleatum-TIGIT interaction is capable of neutralising NK cell cytoxicity. A particular line of mutated NK cells were used for this experimentation. The NK cell mutants are deficient of all inhibitory receptors and thus their cytotoxic activity cannot be neutralised. Unsurprisingly, the mutant NK cells (∆NK) showed no signs of cytotoxic impairment when incubated with F.nucleatum exposed tumours[4]. Next they took the inhibitory receptor deficient ∆NK cells and restored their TIGIT receptor. With a fully functional TIGIT gene the ∆NK cells were equipped with a solitary mechanism for killing inhibition. If the cytotoxicity of these new ∆NK cells (∆NKTIGIT) was lost in presence of F.nucleatum then it had to be via an interaction with TIGIT. This is exactly what they saw [4].

With this confirmation the search began for the bacterial protein responsible for activating TIGIT. An F.nucleatum mutant library was created using transposon mutagenesis technology. Transposons are naturally occurring stretches of DNA that can move around the genome. In the process of relocation transposons can disrupt genes. Utilising this trait allows thousands of mutant bacteria to be created at so that each colony randomly contains a different disruption[11]. A good library of mutants will have at least one disruption mutant for every gene. All of their F.nucleatum mutants were exposed to tumour cells separately. These prexposed tumour cells were then incubated with ∆NKTIGIT cells. Only two mutant strains were unable to inhibit the ∆NKTIGIT cells from killing tumour cells. This implies the disrupted genes in these mutants are required for F.nucleatum’s TIGIT interaction. Fortunately for simplicity both mutants had disruptions to the same gene; Fap2[4]. Thanks to these experiments it has been established that F.nucleatum’s outer membrane protein, Fap2, is capable of localised inhibition to the cytotoxic activity of NK cells (Figure 1).


Figure 1: NK cell cytotoxicity when exposed to wild type and mutant strains of F.nucleatum[4].

Due to this newly found trait it is obvious that F.nucleatum can provide a profound survival advantage to developing tumours. F.nucleatum cannot cause a systemic shut down of NK cell killing. NK cells have to have come in physical contact with the bacterium if they are to lose their cytotoxic capabilities. For a tumour to benefit from this survival advantage the bacterium has to inhabit its tissues. Due to the anoxic conditions in which the bacterium evolved to thrive, it is likely that F.nucleatum is capable of surviving in many different tumour types. Why then has F.nucleatum only really been found with any consistency in colon cancers? The answer is probably a simple matter of exposure. The ease at which F.nucleatum could passage from its native oral environment, through the GI tract, to colonic tumours may explain its prevalence in such carcinomas[4]. The authors highlight that F.nucleatum is an important player in the development and survival of colon cancers, but its role in other cancers remain uncertain. More data is required to fully assess F.nucleatum’s significance to other solid tumours. An interesting question that I would like to see answered is whether the administering of antibiotics alongside the standard treatments for colon cancer can better patients’ prognosis? Obviously this would have to be rigorously tested before any new combination therapies are put in place, but an interesting line of investigation no doubt.

  1. SCOTTING, P. 2010. Cancer A Beginner’s Guide. Oneworld Publications
  2. KOCH, J., STEINLE, A., WATZL, C. & MANDELBOIM, O. 2013. Activating natural cytotoxicity receptors of natural killer cells in cancer and infection.Trends in Immunology, 34, 182-191
  3. COOPER, G,. M. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Signaling Molecules and Their Receptors.Available from:
    (Accessed 20/03/15)
  4. GUR, C., IBRAHIM, Y., ISAACSON, B., YAMIN, R., ABED, J., GAMLIEL, M., ENK, J., BAR-ON, Y., STANIETSKY-KAYNAN, N., COPPENHAGEN-GLAZER, S., SHUSSMAN, N., ALMOGY, G., CUAPIO, A., HOFER, E., MEVORACH, D., TABIB, A., ORTENBERG, R., MARKEL, G., MIKLIC, K., JONJIC, S., BRENNAN, C. A., GARRETT, W. S., BACHRACH, G. & MANDELBOIM, O. 2015. Binding of the Fap2 Protein of Fusobacterium nucleatum to Human Inhibitory Receptor TIGIT Protects Tumors from Immune Cell Attack.Immunity, 42, 344-355.
  5. HAN, Y. P. W., REDLINE, R. W., LI, M., YIN, L. H., HILL, G. B. & MCCORMICK, T. S. 2004. Fusobacterium nucleatum induces premature and term stillbirths in pregnant mice: Implication of oral bacteria in preterm birth.Infection and Immunity, 72, 2272-2279.
  6. KOSTIC, A. D., GEVERS, D., PEDAMALLU, C. S., MICHAUD, M., DUKE, F., EARL, A. M., OJESINA, A. I., JUNG, J., BASS, A. J., TABERNERO, J., BASELGA, J., LIU, C., SHIVDASANI, R. A., OGINO, S., BIRREN, B. W., HUTTENHOWER, C., GARRETT, W. S. & MEYERSON, M. 2012. Genomic analysis identifies association of Fusobacterium with colorectal carcinoma.Genome Research,22, 292-298.
  7. LIU, H., REDLINE, R. W. & HAN, Y. W. 2007. Fusobacterium nucleatum induces fetal death in mice via stimulation of TLR4-mediated placental inflammatory response (vol 179, pg 2501, 2007).Journal of Immunology, 179, 5605-5605.
  8. CHAUSHU, S., WILENSKY, A., GUR, C., SHAPIRA, L., ELBOIM, M., HALFTEK, G., POLAK, D., ACHDOUT, H., BACHRACH, G. & MANDELBOIM, O. 2012. Direct Recognition of Fusobacterium nucleatum by the NK Cell Natural Cytotoxicity Receptor NKp46 Aggravates Periodontal Disease.Plos Pathogens,
  9. STANIETSKY, N., ROVIS, T. L., GLASNER, A., SEIDEL, E., TSUKERMAN, P., YAMIN, R., ENK, J., JONJIC, S. & MANDELBOIM, O. 2013. Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR.European Journal of Immunology, 43, 2138-2150.
  10. STANIETSKY, N., SIMIC, H., ARAPOVIC, J., TOPORIK, A., LEVY, O., NOVIK, A., LEVINE, Z., BEIMAN, M., DASSA, L., ACHDOUT, H., STERN-GINOSSAR, N., TSUKERMAN, P., JONJIC, S. & MANDELBOIM, O. 2009. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity.Proceedings of the National Academy of Sciences of the United States of America, 106,17858-17863.
  11. PRAY, L. 2008.Transposons: The jumping genes. Nature Education 1(1):204
    (Accessed 23/03/15)

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