Enterohemorrhagic Escherichia coli infection stimulates Shiga toxin 1 macropinocytosis and transcytosis across intestinal epithelial cells

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
AJP Cell Physiology (Impact Factor: 3.78). 08/2011; 301(5):C1140-9. DOI: 10.1152/ajpcell.00036.2011
Source: PubMed


Gastrointestinal infection with Shiga toxins producing enterohemorrhagic Escherichia coli causes the spectrum of gastrointestinal and systemic complications, including hemorrhagic colitis and hemolytic uremic syndrome, which is fatal in ∼10% of patients. However, the molecular mechanisms of Stx endocytosis by enterocytes and the toxins cross the intestinal epithelium are largely uncharacterized. We have studied Shiga toxin 1 entry into enterohemorrhagic E. coli-infected intestinal epithelial cells and found that bacteria stimulate Shiga toxin 1 macropinocytosis through actin remodeling. This enterohemorrhagic E. coli-caused macropinocytosis occurs through a nonmuscle myosin II and cell division control 42 (Cdc42)-dependent mechanism. Macropinocytosis of Shiga toxin 1 is followed by its transcytosis to the basolateral environment, a step that is necessary for its systemic spread. Inhibition of Shiga toxin 1 macropinocytosis significantly decreases toxin uptake by intestinal epithelial cells and in this way provides an attractive, antibiotic-independent strategy for prevention of the harmful consequences of enterohemorrhagic E. coli infection.

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Available from: Michael Delannoy, Sep 23, 2015
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    • "On the other hand, if transepithelial or transendothelial CPP-mediated transport of a cargo drug is intended, the prominent criterion for efficient drug delivery relates to whether transcytosis follows the endocytic uptake. Transcytosis is exploited by various toxins subsequent to oral ingestion in order to transverse the epithelium for gaining access to the systemic circulation [87,88], but may also be the mechanism by which peptide and protein drugs are translocated across epithelia and endothelia when CPPs are employed as delivery vectors. As opposed to mediating endocytic cellular uptake, some CPPs are able to directly translocate across the plasma membrane in an energy-independent manner as demonstrated by the fact that CPP-uptake takes place when the temperature is lowered to 4 ˝ C [89]. "
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    ABSTRACT: The hydrophilic nature of peptides and proteins renders them impermeable to cell membranes. Thus, in order to successfully deliver peptide and protein-based therapeutics across the plasma membrane or epithelial and endothelial barriers, a permeation enhancing strategy must be employed. Cell-penetrating peptides (CPPs) constitute a promising tool and have shown applications for peptide and protein delivery into cells as well as across various epithelia and the blood-brain barrier (BBB). CPP-mediated delivery of peptides and proteins may be pursued via covalent conjugation of the CPP to the cargo peptide or protein or via physical complexation obtained by simple bulk-mixing of the CPP with its cargo. Both approaches have their pros and cons, and which is the better choice likely relates to the physicochemical properties of the CPP and its cargo as well as the route of administration, the specific barrier and the target cell. Besides the physical barrier, a metabolic barrier must be taken into consideration when applying peptide-based delivery vectors, such as the CPPs, and stability-enhancing strategies are commonly employed to prolong the CPP half-life. The mechanisms by which CPPs translocate cell membranes are believed to involve both endocytosis and direct translocation, but are still widely investigated and discussed. The fact that multiple factors influence the mechanisms responsible for cellular CPP internalization and the lack of sensitive methods for detection of the CPP, and in some cases the cargo, further complicates the design and conduction of conclusive mechanistic studies.
    Preview · Article · Jan 2016 · International Journal of Molecular Sciences
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    • "Recent studies have demonstrated STEC-induced Stx uptake by macropinocytosis (Lukyanenko et al., 2011; In et al., 2013). To determine whether this pathway was also involved in Stx translocation in our experimental system, we investigated several indicators of macropinocytosis. "
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    ABSTRACT: Haemolytic uraemic syndrome caused by Shiga toxin-producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx-related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarised human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonisation, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx-related events and enhance Stx absorption during STEC infection.
    Full-text · Article · Feb 2014 · Cellular Microbiology
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    • "The generation and accumulation of these vesicles have introduced a watery content into the cytoplasm, which contributed, at least partially, to the steep drop in the refractive index of EPEC-wt-infected cells (Figures 1D&2A). It is worth noting in this context that epithelial cell infection by the related pathogen enterohemorrhagic E. coli has also been reported to induce macropinocytosis [40], which is important for inserting a shiga-like toxin secreted by the microbe into its host. However, the role of EPEC-elicited basolateral (macro)pinocytosis remains to be elucidated. "
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    ABSTRACT: Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.
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