Host cell damage induced by porcine ETEC

Enterotoxigenic Escherichia coli modulates host intestinal cell membrane asymmetry and metabolic activity

Dr. Philip Hardwidge of university of Kansas Medical Center has been working on several types of E. coli, that cause diarrhea and malnutrition in humans and livestock, for quite some time. In this particular article published in 2008 in infection and immunity, he and his team tries to find out the extent to which enterotoxigenic E. coli (ETEC) damages host cells.

ETEC is a common cause of traveler’s and postweaning diarrhea in humans and swine, respectively. ETEC releases several enterotoxins, which lead to water and electrolyte loss from the intestine. These toxins include heat labile enterotoxin (LT), the heat stable enterotoxin (ST) and the enteroaggregative E. coli heat stable enterotoxin 1. It has been earlier observed that several bacteria e.g., Salmonella induce apoptosis of host cells to promote their survival and dissemination. The same has been shown by several serotypes of E. coli and toxins; however, whether ETEC also induces apoptosis of host cell is not known.

Keeping in view of all this information, the work was conceptualized with following objectives:
1. to quantify the ability of ETEC and LT in mediating damage of porcine intestinal epithelial cells,
2. to clarify the role of LT in these processes, and
3. to measure any resultant increases in ETEC adherence.
A summary of results obtained is as follows:

ETEC induces an increase in phosphatidylserine (PS) exposure:
Phosphatidylserine (abbreviated Ptd-L-Ser or PS) is a phospholipid component, usually kept on the inner-leaflet (the cytosolic side) of cell membranes by an enzyme called flippase. When a cell undergoes apoptotic cell death phosphatidylserine is no longer restricted to the cytosolic part of the membrane, but becomes exposed on the surface of the cell.

IPEC-J2 cells were infected with wild type ETEC, a mutant strain deficient in LT expression and a mutant complemented by plasmid based eltAB expression. PS exposure was determined by annexin V staining and cell death was determined by PI staining.
Infection with wt significantly increased the PS exposure while insignificantly increased the cell death compared to that of uninfected cells. Infection with both the mutants also increased the PS exposure to same exten; it also increased cell death. Administration of LT only, modestly increased the PS exposure. Infection with a strain (G58-1) that lacks any known plasmids, produced no enterotoxins and is considered to be a commensal, did not significantly change the PS exposure. Further, the authors incubated host cells with outer membrane vesicles (OMVs) purified from ETEC possessing or lacking LT. It was observed that incubation with OMVs significantly increased the PS exposure irrespective of the presence of LT. however, no significant increase in cell death was observed due to purified OMVs or LTs.

All these results indicate that ETEC causes alterations in the membrane structure of intestinal epithelial cells. These alterations are the ones which are commonly associated with apoptosis. The results also indicate that LT is unlikely the primary determinant of this activity.

ETEC inhibits host calcein-AM degradation
To determine if ETEC also inhibits or alters metabolic activities of the host cells, the investigators infected host cells with different ETEC strains (i.e., wild type, mutants and G58-1) and then incubated the infected cells with calcein-AM. Calcein-AM is a non-fluorescent lipophillic ester that penetrates the cell membrane. Inside the cell, cytosolic esterases act on this calcein-AM and generate calcein, which is a fluorescent non-membrane permeable molecule. Thus, quantification of calcein fluorescence after uptake of calcein-AM provides a convenient way to assess cell viability and metabolic activity. It was observed that the conversion of calcein-AM in ETEC infected cells was significantly inhibited in comparison uninfected cells.
The authors also observed that although a large number of cells failed to stain with PI (that is they were live) they had very limited fluorescence. The authors gated these cells to quantify percent of calcein fluorescence negative cells among only live cells. They observed that 4% of live, uninfected cells had negative calcein fluorescence, while 62% of infected cells had negative calcein fluorescence. Infection with both the mutant strains also significantly inhibited calcein fluorescence. To determine if host calcein fluorescence would recover following removal of ETEC, the investigators treated the host cells with gentamycin and then quantified calcein fluorescence as a function of time after gentamycin treatment and they observed that percentage of calcein negative cells decreased from 62% to 14.3% over 1hr following the addition of gentamycin. The results of these experiments suggested that the metabolic activity of the host recovers rapidly after removal of ETEC.
All together, these data indicate that porcine ETEC profoundly alters the membrane asymmetry and metabolic activity of infected host cells.

ETEC does not induce host DNA double-strand breaks:
Double stranded DNA breaks are known to occur in late stages of apoptosis. To determine whether ETEC also causes such breaks, the investigators employed TUNEL assay. No significant difference in TUNEL staining was observed as a result of ETEC infection. These data indicate that while ETEC may induce changes to the host cell which are associated with early stages of apoptosis, e.g., alteration in membrane structure, subsequent signal transduction events mat be inhibited at a later time point in the pathway.

Diversity in porcine ETEC propensity to induce host cell damage:
The authors next used 11 other, different ETEC strains to infect host cells. The results of PS exposure were:
--Three strains induced PS exposure in more than 20% of cells
--Two strains induced PS exposure in 10 to 15% of cells
--Remaining six strains did not induce significant PS exposure
The results of calcein fluorescence experiments were
--Four strains reduced host calcein fluorescence levels equivalent to that observed with the earlier wt strain.
--Three strains has an intermediate phenotype.
--Remaining four strains did not induced any significant decrease in calcein fluorescence.

Furthermore, the strains that induced PS exposure and reduced calcein fluorescence, except one, are known to display either K88a or F41 fimbrae and efficiently bind to the cell lines used in these experiments.
The strains that failed to induce changes in PS exposure or host calcein fluorescence levels are known to display F18 or unknown fimbria phenotypes yet still possess various enterotoxins. These observations lead to the idea that the ETEC enterotoxins are not significant in mediating early apoptotic changes and an important step in causing apoptosis is adherence to host cells.

Host cell damage increase ETEC adherence:

The authors then tested whether caspases are activated in response to ETEC infection, as caspases play important role in apoptosis of cells. They found that both the wild type and mutant strains activated host caspase 3, whereas infection with LT or G58-1 did not activated the caspase 3.
The authors next hypothesized that inducing host cell apoptosis would increase host cell adherence. To test this, cells were treated with camptothecin (promotes apoptosis) and then with ETEC and subsequent adherence was determined. It was observed that pretreatment of host cell with camptothecin significantly increase adherence of ETEC to host cells. Importantly, this increase was observed in both wild type and mutant strains. These results suggested that increase in adherence is independent of the LT expression. To further determine if increase in adherence was dependent upon the activation of host caspases, the authors co-treated the host cells with camptothecin and Ac-DEVD-CHO, an inhibitor of caspase3 dependent pathways. It was observed that Ac-DEVD-CHO caused the adherence to decrease to basal levels.
Taken together all these data indicate that inducing apoptosis in host cells is sufficient to promote subsequent ETEC adherence and that this phenomena require the activity of caspase 3 and/or the downstream effectors of this enzyme.
Furthermore, to assess whether any factor is secreted by apoptic cells in response to chemical induction of apoptosis that might be sensed by ETEC and helps to promote adherence, the authors conducted an experiment. They treated host cells with camptothecin and then treated one set of cells with Ac-DEVD-CHO. They then filtered the supernatant from these cells and added it to culture of ETEC. After 24hrs of incubation, these ETEC inocula were used to infect host cells. It was observed that presence of both camptothecin and Ac-DEVD-CHO abolished the adherence promoting ability of ETEc. Thus, it was possible that a factor released from host cells after activation of caspase 3 apoptic pathways may play a role in regulation of gene expression of pathways associated with ETEC adherence.