Bacterial genotoxins : an interface between infection and cancer biology

Abstract: Infections contribute to the development of more than 20% of all malignancies in humans. While strong evidence exists for a number of virus-associated cancers, the role of bacterial infections is still poorly appreciated although there are evidences that link chronic bacterial infections to increased risk of certain tumors. In the context of bacteria-induced carcinogenesis, bacterial genotoxins might contribute to increase the occurrence of genomic instability during chronic infection. These toxins are produced by several Gram-negative bacteria and are unique among other bacterial toxins since they are able to reach the nucleus and cause DNA damage in intoxicated cells. In this thesis we have investigated the contribution of two bacterial genotoxins to tumor development in acute and chronic intoxication, and their role in the context of chronic infections, using in vivo and in vitro models. We have used as a model the Cytolethal Distending Toxin (CDT) produced by several Gram-negative bacteria and the Typhoid Toxin (TT) produced by Salmonella enterica serovar Typhi. In the context of acute intoxication, CDT-induced DNA damage triggers the activation of the integrin β1, increase of focal adhesions formation and cell spreading in epithelial cells during short-term intoxication experiments (Paper I). This phenotype is mediated by an inside-out integrin signaling and is drastically reduced when the actin cytoskeleton remodeler NET1 is knocked down. We have also demonstrated that two components of the endosomal sorting complex required for transport (ESCRT) known to regulate integrin intracellular trafficking, ALIX and TSG101, are important for the anchorage-independent survival induced by sustain integrin activation. Chronic intoxication with CDT leads to increased mutation frequency, accumulation of chromosomal aberrations and enhanced anchorage-independent growth in vitro (PaperII). Cells treated with sub-lethal doses of CDT during a period of 220 days showed an altered DNA damage response and a higher activation of the MAP kinase p38 signalling, which is known to play an important role in cell survival during acute CDT intoxication. In order to investigate the role of genotoxins in vivo and their carcinogenic potential we designed a new Salmonella infection model in mice where we used a S. enterica serovar Typhimurium (S. Typhimurium) strain able to produce the Typhoid Toxin and, as a control, an isogenic strain that expressed an inactive Typhoid Toxin (PaperIII). We have infected immunocompetent sv129 mice for up to 6 months and the presence of an active version of the toxin led to a reduced mortality rate and higher persistence of the bacteria in some organs suggesting its role in the establishment of a chronic infection. Mice infected with the toxigenic S. Typhimurium strain also showed an altered inflammatory response in intestine and liver and a more pronounced intestinal microbiota alteration compare to the mice infected with the non-toxigenic strain. To better understand how the TT is delivered to the host cells we further investigate its secretion and internalization mechanisms in vitro (Paper IV). We have demonstrated that the Typhoid Toxin is loaded into outer membrane vesicles (OMVs) and is secreted by S. Typhimurium bacteria in the Salmonella containing vacuole (SCV). Vesicles are released by exocytosis-like process in the extracellular space, where they can deliver the genotoxic component to bystander cells. Our data give new important contributions in the studying of bacterial genotoxins and their role in chronic bacterial infection and cancer development