Also and in distinction to intact EHEC [twenty,forty four], the EHEC-L did not lessen the TER of T84 monolayers at any experimental time level (up to 24 h) and the TER of one mg/mL EHEC-L-treated monolayer (one,986 205 m2) was indistinguishable from the management monolayers (2,058 167 m2, n=48 monolayers for every condition, p < 0.001), further suggesting that transcytosis of the cargo occurs via a transcellular and not paracellular pathway. Also, TEM indicates that EHEC-L-induced apical blebbing is not the hallmark of massive cell death. Taken together, these data indicate that bacterial factors, through actin remodeling, induce a novel pathway for transepithelial delivery of Stx1 and Stx2 and possibly other antigens from the apical to basolateral side of the intestinal epithelium.It has been previously reported [42,45] that serine protease autotransporters of Enterobacteriaceae (SPATEs), namely Pet (plasmid-encoded toxin) from EAEC and EspC (E. coli secreted Figure 7. EHEC-L induced MPC leads to the transcellular transcytosis of the apical cargo. (A)152121-30-7 Representative TEM image of T84 cells treated apically for 4h with a mixture of EHEC-L and 1 mg/mL HRP. EHEC-L causes the formation of macropinosomes filled with HRP (black arrowheads). (B) Representative TEM image depictures the process of a formation of HRP-bearing macropinosomes (black arrowhead). The apical EHEC-L induced bleb (white arrowhead) upon retraction back into the cell [19] and closure forms a new HRP-containing macropinosome. (C) Representative TEM image shows that the HRP-bearing macropinosome is reaching the basolateral side of filter-grown T84 cells (white arrow) and makes contact with the basal membrane. (D) Representative image obtained from fluorescence plate reader shows that EHEC-L stimulates Stx1 transcytosis in a time-dependent manner. This transcytosis is significantly inhibited by cytD (Table 2)from enteropathogenic E. coli (EPEC) strains, possess a consensus serine protease motif that causes actin remodeling in IEC. Many EHEC strains including EDL933 also express a SPATE family member termed EspP (E. coli secreted protein P). Thus, we tested a hypothesis that EspP might be responsible for stimulation of MPC in T84 cells. We took advantage of a previously reported laboratory strain E. coli K-12 transformed with the espP gene [46]. T84 cells were treated with lysates (0.3 mg/mL) prepared from either K-12EspP strain (EspP-L), parental K-12 strain (K-12-L) that naturally lacks EspP, or EHEC-L, each in the presence of Stx1 for 4 h. The amount of endocytosed Stx1 was measured in total cell lysates (Figure 8A). EspP-L was sufficient to stimulate Stx1 uptake compared to K-12-L or control cells not exposed to bacterial lysates. Moreover, the amount of Stx1 internalized by EspP-L-treated T84 cells was similar to cells treated with EHEC-L. Surprisingly, EspP was also internalized by T84 cells and the amount of endocytosed Stx1 correlated with the amount of EspP in T84 cell lysates. This EspP-induced increase in Stx1 uptake was accompanied by significant actin remodeling (Figure 8B) with toxin residing inside actin-coated macropinosomes, similar to what we have detected in T84 cells results in the transepithelial delivery of macropinocytosed material from the mucosal to serosal side. In conclusion, EHEC-induced actin remodeling that is necessary for Stx MPC and transcytosis does not require active EspA-mediated type 3 secretion or intimin-mediated attachment, and is different from mechanisms of actin remodeling involved in pedestal formation. EHEC soluble factor(s), particularly serine protease EspP, is sufficient to stimulate Stx MPC and transcellular transcytosis in vitro and in vivo. Importantly, soluble factor(s) from another deadly enteric pathogen, EAEC H104:O4, is also able to stimulate a similar pathway leading to significant increase in Stx uptake.The 2011 outbreak of STEC diseases started in Germany, spread through 16 countries and underscored the public health importance of this type of foodborne pathogen [12,13]. Several features make STEC particularly worrisome. New extremely virulent STEC strains different from classical EHEC O157:H7 are evolving [1,6,7]. The number of EHEC-related outbreaks has increased markedly in recent years worldwide along with an increase in economic burden and deaths. Once they are established, there are no effective treatments for intestinal or systemic STEC illnesses [4,6]. Antibiotics given for STECrelated diarrhea, particularly those that target bacterial DNA, increase the risk of developing HUS [4,47]. Soluble multivalent Gb3 receptor-based Stx1 and Stx2 binding agents [6,16] did not succeed as an anti-toxin treatment when administered in the intestine. Better characterization of the molecular mechanisms of Stx1 and Stx2 uptake and transcytosis by human enterocytes, the gateway to systemic dissemination of these toxins, could identify targets for novel therapeutic approaches for STEC diseases. The present study provides insights into the molecular mechanism of Stx1 and Stx2 uptake by human enterocytes in the absence of Gb3 receptors and examines transcytosis across the intestinal epithelial barrier at the earliest stage of EHEC infection, ahead of significant ischemia and inflammation. Our current data suggest that EHEC infection stimulates toxin endocytosis and transcytosis by enterocytes, initiating the actin remodeling that leads to toxin MPC. This actin rearrangement necessary for toxin MPC and transcellular transcytosis is independent of type 3 secretion and intimin attachment. Several lines of evidence indicate that formation of actin pedestals and macropinosomes occur by two distinct actin polymerization-depolymerization pathways orchestrated by EHEC that serve different goals in EHEC pathogenesis. The end point of T3SS-mediated actin remodeling is the anchoring of the bacteria to the apical surface of enterocytes. The result of MPC is a transfer of high molecular weight luminal cargo, including Shiga toxins, from the mucosal to the serosal side. Intact EHEC, while required for pedestal formation, are not necessary to stimulate MPC. Bacterial soluble factor(s) present in lysates of EHEC or EAEC is sufficient to carry out this actin rearrangement. Stimulation of MPC by bacterial lysates in vitro and in vivo results in significantly increased Stx1 and Stx2 endocytosis.Fluorescence intensity (A.U.) of transcytosed Stx1, HRP or dextran normalized to background fluorescence intensity in control and experimental conditions significant versus corresponding controls NS not significant versus corresponding controls n – number of monolayers treated either with EHEC [20], EHEC-L, or EAEC-L. These data indicate that EHEC-expressed serine protease EspP is capable of stimulating Stx1 uptake in IEC through actin remodeling and formation of macropinosomes in vitro. Consequently, we tested whether EspP-L stimulates MPC and possibly transcytosis of macropinocytic cargo in vivo. Because multiphoton microscopy resolution (Figure 5C) was not sufficient to resolve the intracellular vesicles inside the mouse enterocytes (due in part to tissue autofluorescence, light scattering, and smaller cell size in tissue compared to cultured T84 cells), we applied TEM to detect EspP-induced changes in tissue. For these experiments, two 1 cm loops were created in each mouse (n = 2 animals). Loops were injected with 2 mg/mL HRP and either 1 mg/mL K-12-L (control) or 1 mg/mL EspP-L (experimental). To detect possible HRP transcytosis into the lamina propria, we extended the experimental time to 6 hours, after which mice were sacrificed and tissue fixed and prepared to detect HRP by TEM. EspP induced the appearance of macropinosomes which varied in size and shape (Figure 8C). A number of macropinosomes carried HRP inside. HRP-bearing vesicles were often concentrated near basolateral membranes, a potential site of HRP transcytosis. Importantly, HRP was readily detectable in the submucosa, demonstrating that EspPL treatment leads to transepithelial trafficking of luminal macropinocytic cargo and release of cargo (in this case HRP) into the lamina propria in mouse ileum. All mentioned observations, including macropinosomes (empty or HRPbearing) and HRP transcytosis were not detected in control samples. EspP treatment also damaged the brush border of mouse enterocytes compared to the control mouse enterocytes (Figure 8C). We conclude that EspP is sufficient to trigger macropinocytosis of high molecular weight cargo in vivo, which Figure 8. Serine protease EspP is sufficient to stimulate Stx1 MPC in T84 cells. (A) Representative IB and quantitative representations of IB data show that EspP expression by bacteria is sufficient to significantly increase Stx1 uptake in T84 cells compared to untreated cells or cells treated with lysates from K-12 bacteria that do not express EspP (n 3 monolayers per experimental condition – significant compared to the control (p < 0.05)). (B) Representative confocal optical sections through T84 cells incubated for 4 h in the presence of EspP-L (1 mg/mL) and B-subunit of Stx (Stx1B 0.5 /mL) show that EspP reorganized actin in T84 cells and triggered the formation of actin coated macropinosomes that filled with Stx1B. F-actin - red by phalloidin-AlexaFluor568 Stx1B - green by AlexaFluor 488. (C) Representative TEM images of mouse ileal tissue treated from the luminal side with either a mixture of EspP-L and 2 mg/mL HRP or with mixture of K-12-L and 2 mg/mL HRP (control), bar -2 . EspP-L caused the formation of macropinosomes (black arrowheads) often containing HRP (black vesicles inside the macropinosomes). The macropinosomes are completely absent from control tissue. Importantly, EspP-L treatment caused the HRP accumulation in lamina propria (white arrows), which indicates the HRP transepithelial delivery. In contrast, HRP was absent from lamina propria (white arrows) in control tissue. Macropinosomes were often concentrated close to the lateral membranes (small black arrows) in ileal tissue, similar to observations in T84 cells, bars -2 .The molecular mechanisms of MPC and pedestal formation are substantially different. The molecular events involved in T3SS- and intimin-dependent EHEC attachment to IEC are well characterized [257]. By contrast, the mechanisms of MPC, particularly EHEC-stimulated MPC, are just emerging [20,21,24]. Comparison of effects of EHEC lysates versus intact bacteria has allowed us to begin to dissect the molecular signaling cascade necessary for toxin MPC from other aspects of bacteria-host interaction. The two processes are actindependent both are inhibited by the actin-depolymerizing drug cytD [38]. However, the formation of actin pedestals requires cortactin, which is recruited by T3SS effectors to the site of bacterial attachment [37,38]. In contrast, MPC does not require cortactin and cortactin is absent from the macropinocytic blebs. The ATP-dependent motor protein NMIIA is necessary for MPC, as inhibition of NMIIA activity by drugs, shRNA [20] or MLC inhibition substantially reduces toxin MPC. Thorough analysis of cytoskeletal proteins in EHEC pedestals did not reveal NMIIA. Instead the actin binding protein tropomyosin was recruited to the sites of active actin rearrangement in these pedestals by Tir, a T3SS effector [48]. These data suggest that enteric pathogens such as EHEC that reorganize the host actin cytoskeleton during the course of infection may affect it in several ways. Some actin reorganization is pathogen-specific, allowing particular bacteria to gain the advantage in colonization, as in the case of characteristic EHEC attaching and effacing lesions. Others, including actin-dependent MPC, are less specific and likely shared among several groups of enteric pathogens. Our data showing that lysates from intiminnegative Stx-producing strain of EAEC also cause formation of apical MPC and stimulate Stx uptake by IEC strongly supports this suggestion. These data are also in good agreement with previously published observations that EAEC infection of T84 cells causes damage of microvilli, blebbing of apical membrane and the appearance of multiple large vacuoles in the cytoplasm of affected cells [42]. These morphological changes caused by intact EAEC are very similar to what we have observed in cells treated with intact EHEC, or EHEC-L, or EAEC-L, or EspP-L and represent MPC. Importantly, these MPC-induced morphological changes do not represent a massive cell death because the TER, which serves as an indicator of intestinal barrier function, does not decrease upon EHEC-L treatment and is similar to that in control monolayers not exposed to EHEC-L. Analysis of EHEC soluble factors secreted independently of T3SS which might be involved in host actin remodeling suggested that serine protease EspP may be responsible for triggering MPC [42,45,46]. Indeed, lysates from EspPexpressing K-12, but not from the isogenic K-12 strain, are able to increase Stx1 uptake in IEC similar to EHEC-L and EAEC-L. This EspP-induced increase in toxin endocytosis was accompanied by significant actin remodeling, and toxin was carried into the cells and across the cells by the actin-coated macropinosomes. Importantly, similar serine proteases termed Pet are secreted by EAEC strains [42,45,49] and might be responsible for triggering the observed toxin macropinocytosis in H104:O4-induced disease. MPC is not cargo-specific endocytosis, as indicated by uptake of Stx1 and Stx2 as well as HRP and dextran. These data suggest that high molecular weight bacterial products other than the toxins might successfully use this pathway to get inside the enterocytes. Importantly, data from us and others indicate that MPC might serve as a mechanism for movement of cargo from the intestinal lumen to the serosa while avoiding lysosomal or proteosomal degradation [20,50]. 23551948This newly recognized, actin-dependent transcellular transcytosis may represent an early antigen-presenting pathway in the intestine before TJ permeability is compromised by inflammation or other factors, and it may potentially be a major route for the systemic delivery of Stx1 and Stx2 at the earliest stages of infection. Thus, the identification of molecular targets to inhibit Stx MPC by IEC may prevent not only Stx-induced intestinal problems but also systemic complications from STEC. The mechanisms of interaction of Stx1 and Stx2 with different types of IEC are likely different. It has been shown that Paneth cells in human small intestine bind Stx1, but not Stx2, via a proposed receptor-mediated pathway [51], although the role of Paneth cells in Stx-induced disease remains uncharacterized [7]. It has also been recently shown that Mcells, which specialize in transcellular transcytosis of antigens,can transcellularly transport not only the toxins, but also the intact EHEC [40].
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