The rapid progress in diverse approaches and technologies such as genome sequencing, gene mutation, site-directed mutagenesis, proteomics, crystallography, high-throughput screening and cryo-electron microscopy has provided unprecedented knowledge regarding novel functions during E. coli pathogenesis. In this chapter, we discuss six novel mechanisms of virulence and pathogenesis into the different E. coli pathotypes. These mechanisms include two nanomachines (T3SS and T6SS) used to colonize and hijack the host cell functions (T3SS, T6SS) or for bacterial competition (T6SS), as well as the mechanisms of T3SS protein-protein interaction, which has allowed the development of blocking compounds or peptides as preventing strategies; likewise, mechanisms of some pathogenic E. colimicrobiota; and finally, mechanisms to regulate the expression of factors related with the lifestyle of these bacterial pathogens as those forming bacterial communities or new mechanisms of remote interaction among bacteria and host cells, such as the delivery of vesicles containing cargo molecules.