Bacterial pathogens utilize a myriad of mechanisms to invade mammalian hosts, damage tissue sites, and evade the immune system. One essential strategy of Gram-negative bacteria is the secretion of virulence factors through both inner and outer membranes to reach a potential target. Most secretion systems are harbored in mobile elements including transposons, plasmids, pathogenicity islands, and phages, and Escherichia coli is one of the more versatile bacteria adopting this genetic information by horizontal gene transfer. Additionally, E. coli is a bacterial species with members of the commensal intestinal microbiota and pathogens associated with numerous types of infections such as intestinal, urinary, and systemic in humans and other animals. T6SS cluster plasticity suggests evolutionarily divergent systems were acquired horizontally. T6SS is a secretion nanomachine that is extended through the bacterial double membrane; from this apparatus, substrates are conveyed straight from the cytoplasm of the bacterium into a target cell or to the extracellular space. This nanomachine consists of three main complexes: proteins in the inner membrane that are T4SS component-like, the baseplate complex, and the tail complex, which are formed by components evolutionarily related to contractile bacteriophage tails. Advances in the T6SS understanding include the functional and structural characterization of at least 13 subunits (so-called core components), which are thought to comprise the minimal apparatus. So far, the main role of T6SS is on bacterial competition by using it to kill neighboring non-immune bacteria for which antibacterial proteins are secreted directly into the periplasm of the bacterial target after cell–cell contact. Interestingly, a few T6SSs have been associated directly to pathogenesis, e.g., roles in biofilm formation and macrophage survival. Here, we focus on the advances on T6SS from the perspective of E. coli pathotypes with emphasis in the secretion apparatus architecture, the mechanisms of pathogenicity of effector proteins, and the events of lateral gene transfer that led to its spread.

Until now, a common feature that defines the enteroaggregative Escherichia coli (EAEC) strains is the ability to produce a ‘stacked-brick’ appearance

A hallmark of enteroaggregativeEscherichia coli(EAEC) infection is a formation of biofilm, which comprises a mucus layer with immersed bacteria in the intestines of patients. While studying the mucinolytic activity of Pic in anin vivosystem, rat ileal loops, we surprisingly found that EAEC induced hypersecretion of mucus, which was accompanied by an increase in the number of mucus-containing goblet cells. Interestingly, an isogenicpicmutant (EAEC Δpic) was unable to cause this mucus hypersecretion. Furthermore, purified Pic was also able to induce intestinal mucus hypersecretion, and this effect was abolished when Pic was heat denatured. Site-directed mutagenesis of the serine protease catalytic residue of Pic showed that, unlike the mucinolytic activity, secretagogue activity did not depend on this catalytic serine protease motif. Other pathogens harboring thepicgene, such asShigella flexneriand uropathogenicE. coli(UPEC), also showed results similar to those for EAEC, and construction of isogenicpicmutants ofS. flexneriand UPEC confirmed this secretagogue activity. Thus, Pic mucinase is responsible for one of the pathophysiologic features of the diarrhea mediated by EAEC and the mucoid diarrhea induced byS. flexneri.

The intestinal epithelium consists of a single cell layer, which is a critical selectively permeable barrier to both absorb nutrients and avoid the entry of potentially harmful entities, including microorganisms. Epithelial cells are held together by the apical junctional complexes, consisting of adherens junctions and tight junctions (TJs), and by underlying desmosomes. TJs lay in the apical domain of epithelial cells and are mainly composed by transmembrane proteins such as occludin, claudins, JAMs, and tricellulin, that are associated with the cytoplasmic plaque formed by proteins from the MAGUK family, such as ZO-1/2/3, connecting TJ to the actin cytoskeleton, and cingulin and paracingulin connecting TJ to the microtubule network. Extracellular bacteria such as EPEC and EHEC living in the intestinal lumen inject effectors proteins directly from the bacterial cytoplasm to the host cell cytoplasm, where they play a relevant role in the manipulation of the eukaryotic cell functions by modifying or blocking cell signaling pathways. TJ integrity depends on various cell functions such as actin cytoskeleton, microtubule network for vesicular trafficking, membrane integrity, inflammation, and cell survival. EPEC and EHEC effectors target most of these functions. Effectors encoded inside or outside of locus of enterocyte effacement (LEE) disrupt the TJ strands. EPEC and EHEC exploit the TJ dynamics to open this structure, for causing diarrhea. EPEC and EHEC secrete effectors that mimic host proteins to manipulate the signaling pathways, including those related to TJ dynamics. In this review, we focus on the known mechanisms exploited by EPEC and EHEC effectors for causing TJ disruption.

Many pathogenic bacteria subvert normal host cell processes by delivering effector proteins which mimic eukaryotic functions directly into target cells. EspF is a multifunctional protein injected into host cells by attaching and effacing pathogens, but its mechanism of action is not understood completely. In silico analyses of EspF revealed two key motifs: proline-rich domains and PDZ domain binding motifs. Such functional domains may allow EspF to act as an actin nucleation-promoting factor by mimicking host proteins. In agreement with these predictions, we found that EspF from rabbit enteropathogenic Escherichia coli (E22) participates in the regulation of actin polymerization by binding to a complex of proteins at the tight junctions (TJ). EspF bound to actin and profilin throughout the course of infection. However, after 2 h of infection, EspF also bound to the neural Wiskott-Aldrich syndrome protein and to the Arp2/3, zonula occludens-1 (ZO-1), and ZO-2 proteins. Moreover, EspF caused occludin, claudin, ZO-1, and ZO-2 redistribution and loss of transepithelial electrical resistance, suggesting that actin sequestration by EspF may cause local actin depolymerization leading to EspF-induced TJ disruption. Furthermore, EspF caused recruitment of these TJ proteins into the pedestals. An E22 strain lacking EspF did not cause TJ disruption and pedestals were smaller than those induced by the wild-type strain. Additionally, the pedestals were located mainly in the TJ. The overexpression of EspF caused bigger pedestals located along the length of the cells. Thus, actin sequestration by EspF allows the recruitment of junctional proteins into the pedestals, leading to the maturation of actin pedestals and the disruption of paracellular permeability.

EspC ( Escherichia coli secreted protein C) of enteropathogenic E. coli (EPEC) shows the three classical domains of the autotransporter proteins and has a conserved serine protease motif belonging to the SPATE (serine protease autotransporters of Enterobacteriaceae ) subfamily. EspC and its homolog Pet in enteroaggregative E. coli (EAEC) bear the same sequence within the serine protease motif, and both proteins produce enterotoxic effects, suggesting that like Pet, EspC could be internalized to reach and cleave the calmodulin-binding domain of fodrin, causing actin cytoskeleton disruption. Even though both proteins cause cytoskeleton damage by virtue of their serine protease motifs, the following evidence supports the hypothesis that the mechanisms are different. (i) To obtain similar cytotoxic and cytoskeletal effects, a threefold-higher EspC concentration and a twofold-higher exposure time are needed. (ii) EspC internalization into epithelial cells takes more time (6 h) than Pet internalization (30 min), and the distributions of the two proteins inside the cells are also different. (iii) Both proteins have affinity for fodrin and cleave it, but the cleavage sites are different; EspC produces two cleavages, while Pet produces just one. (iv) EspC does not cause fodrin redistribution within epithelial cells. (v) An EspC serine protease motif mutant, but not a Pet serine protease mutant, competes with EspC by blocking cytoskeletal damage. All these data suggest that the protein conformational structure is very important for the activity of the catalytic site, influencing its interaction with the target protein and its internalization. The differences between these proteins may explain the reduced ability of EspC to cause cytopathic effects. However, these differences may confer a specialized role on EspC in the pathogenesis of EPEC, which is different from that of Pet in EAEC pathogenesis.