4B). Together these results show that at the highest dose of meprins, the decreased abilities of AIEC bacteria to adhere to and invade intestinal epithelial cells were not Trichostatin A (TSA) due to degradation of LF82 OMPs or flagella. Figure 4 Proteolytic activity of meprins �� and �� on AIEC LF82 outer membrane proteins and flagellin. Meprins �� and �� induce proteolytic cleavage of AIEC LF82 type 1 pili Experiments performed in vitro with cultured intestinal epithelial cells, ex vivo with human isolated enterocytes from CD patients, or in vivo using transgenic mice expressing the human CEACAM6 receptors showed that type 1 pili play a key role in the ability of AIEC bacteria to adhere to and invade intestinal epithelial cells [12], [13], [15].

We investigated whether the decrease in the abilities of AIEC bacteria to adhere to and invade epithelial intestinal cells observed after pretreatment of bacteria with meprin �� or meprin �� could be the result of proteolytic degradation of type 1 pili by these proteases. A dose-dependent proteolytic degradation of FimA, the major subunit of type 1 pili, was observed after treatment of AIEC LF82 purified type 1 pili with meprins �� and �� (Fig. 5A). Treatment of LF82 purified type 1 pili with 100 ��g/ml of meprin �� or meprin �� induced a strong decrease in the FimA band observed on SDS-PAGE compared to untreated type 1 pili (89% for meprin �� and 72% for meprin ��). We also analyzed the proteolytic activity of meprins on type 1 pili present on the surface of whole bacteria.

The amount of FimA subunit relative to that of the inner membrane protein Lep was determined by Western blot after treatment of LF82 bacteria with active or heat-inactivated meprin �� or ��. We observed 58% and 34% decreased amounts of FimA after treatment of the bacteria with active meprins �� and ��, respectively compared to those of untreated bacteria (Fig. 5B). In contrast, when bacteria were treated with heat-inactivated meprins the amount of FimA was unchanged. The degradation products of purified AIEC LF82 type 1 pili following meprin treatment were analyzed by mass spectrometry. Treatment of purified LF82 type 1 pili with 100 ��g/ml of active meprins �� and ��, compared to heat inactivated meprins, modified the mass spectrometry profile of type 1 pili (Fig. 5C and 5D). A decrease in the intensity of the base peak of spectrum corresponding to the protonated form ([M + H]+) of major analyte detected between 16259 and 16267 m/z was observed after treatment with Entinostat meprins. This was no longer observed with heat inactivated meprins. The same observation was made with the doubly protonated form ([M + 2H]2+) of this analyte, detected between 8130 and 8134 m/z after treatment with active meprins.