Enteropathogenic E.coli Induced Diarrhea
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Enteropathogenic E.coli Induced Diarrhea
Pathogenic microbes subvert normal host-cell processes to create a specialized niche, which enhances their survival. A common and recurring target of pathogens is the host cells cytoskeleton, which is utilized by these microbes for purposes that include attachment, entry into cells, movement within and between cells, vacuole formation and remodeling, and avoidance of phagocytosis. One such subversive microbe is a gram-negative pathogen EPEC (Enteropathogenic Escherichia coli) that causes chronic, watery diarrhea in humans, primarily young children and infants. Central to EPEC-mediated disease is its colonization of the intestinal epithelium. After initial adherence, EPEC causes the localized effacement of microvilli and intimately attaches to the host cell surface, forming characteristic A/E (attaching and effacing) lesions. A/E pathogens typically reside on a pedestal on the surface of the host epithelial cell and ultimately cause severe disruption of the microvilli brush border (Ref.1). All the genes necessary for the formation of A/E lesions by EPEC are contained within a 35-kbp pathogenicity island termed the LEE (Locus of Enterocyte Effacement). These include the Esps (E. coli-secreted protein, EspA, EspB, EspD), Escs (E. coli secretion), Sep (secretion of E. coli proteins), Eae (E. coli attaching and effacing that encodes Intimin), and TIR (Translocated Intimin Receptor) genes. The translocation of EspA, EspB, and EspD proteins is essential for activating a number of signal transduction pathways, although their precise role in pathogenesis is not well defined. All of these effector proteins are secreted by a Type-III secretion system encoded by the Esc and Sep genes (Ref.2).

The host cell undergoes a number of changes during infection by EPEC. The most striking change in the cellular structure of the host cell is the formation of characteristic Actin pedestals. Upon contact with the host cell, EPEC secretes the virulence factor TIR through Type-III secretion system into the cytoplasm of the target cell. TIR is then inserted in the plasma membrane of the host cell and serves as the receptor for the bacterial surface protein intimin, which promotes a dramatic reorganization of the cytoskeleton to form the pedestal upon which EPEC resides. TIR is phosphorylated at its cytoplasmic region by a yet unknown cellular kinase and recruits a host-cell GTPase, which in turn recruits the WASP (Wiskott-Aldrich Syndrome Protein). WASP proteins serve a scaffolding function to bring together Actin monomers and ARP2/3 (Actin-Related Proteins) to form a nucleation core (Ref.3). N-WASP and the ARP2/3 complex are recruited to the pedestal tip, F-Actin, Alpha-Actin, Talin, Ezrin, and Villin are found along the length of the pedestal whereas nonmuscle Myosin-II and tropomyosin are found at the pedestal base. Shifts in intracellular calcium levels also seem to play a role in EPEC pathogenesis. Increases in intracellular calcium levels result in the depolymerization of Actin by Villin and a breakdown of the host cytoskeleton. Activation of PLC-Gamma (Phospholipase-C-Gamma) leads to the formation of IP3 (Inositol triphosphate) and DAG (Diacylglycerol), and IP3 is involved in the release of Ca2+ from intracellular stores. Cytosolic PKC (Protein Kinase-C) also gets activated upon EPEC infection and translocates to the plasma membrane. In addition to the dramatic Actin restructuring leading to pseudopod formation, EPEC also alters the phosphorylation states of several host proteins. Among the proteins prominently phosphorylated upon EPEC infection is MLC (Myosin Light Chain), which is associated with the cytoskeletal fraction of the host cell. MLC is phosphorylated by MLCK (Myosin Light Chain Kinase), which causes contraction of the perijunctional actomyosin ring. This opens tight junctions, presumably by increasing the tension on the tight junction, resulting in a decrease of the transepithelial resistance. The initial phosphorylation event probably occurs via PKC, which is activated upon EPEC infection. These effects on MLC ultimately lead to a disruption of tight junction integrity and an increase in paracellular permeability. Increasing tight junction permeability alters normal ion transport mechanisms, thus allowing electrochemical gradients to reach equilibrium. In addition to this disruption, EPEC alters net ion secretion in polarized epithelial cells. It inhibits net ion secretion stimulated by classic secretagogues, which results in a decreased short circuit current. This attenuation is not from Cl- secretion but may be partly dependent on HCO3- secretion (Ref.4). A rapid effect on ion secretion, perhaps through Cl- or HCO3-, triggers the initial onset of diarrhea and the following cytoskeletal changes and polymorphonuclear leukocytes transmigration contribute to the prolonged disease.

EPEC infection, like many other pathogens also induces an inflammatory response in the host. It stimulates the phosphorylation and degradation of I-KappaB-Alpha, and triggers the NF-KappaB (Nuclear Factor kappaB) subunits p50 and p65. This factor then stimulates the transcription of IL-8 (Interleukin-8), a chemoattractant that functions in recruiting polymorphonuclear leukocytes to infections in the host. During EPEC infection, polymorphonuclear leukocytes transmigrate across the intestinal epithelium from the MALT (Mucosal-Associated Lymphoid Tissue) to the site of bacterial attachment in response to increased IL-8. A myriad of host cell signaling pathways, including MAPK (Mitogen-Activated Protein Kinases), PKCs , tyrosine kinases, and ROIs (Reactive Oxygen Intermediates) contribute to the mucosal inflammatory response associated with EPEC infection. Signals that effectuate the inflammatory response, such as MAPKs, play no role in the permeability changes. Therefore, the many signals that are activated after EPEC attachment appear to diverge and impact separate physiological functions (Ref.5).

Pathogenic E. coli strains remain a leading cause of severe and persistent infant diarrhea in developing countries. EPEC is estimated to kill several hundred thousand children a year worldwide and is the leading cause of bacterially mediated diarrhea in children. EPEC primarily causes chronic watery diarrhea, often accompanied by a low-grade fever and vomiting. Like most diarrheagenic pathogens, EPEC is transmitted via the oral-fecal route. There are other members of the A/E family of pathogens in addition to EPEC. These include Enterohemmorhagic E. coli (EHEC, the causative agent of hemorrhagic colitis and hemolytic uremic syndrome), a human pathogen, and numerous animal pathogens that cause disease in rabbits, pigs, dogs, and mice. These all induce Actin polymerization and pedestal formation in a manner analogous to EPEC (Ref.4).