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The time dependency of in vivo inhibition and labelling was investigated and the results are described in this section. In order to obtain a sufficient amount of bacteria for SDS-PAGE analysis and visualisation at each time point, all treated samples were grown and harvested at the same time with inhibitor added at different stage of the growth curve in each case. Briefly, a 1 hour pre-grown C. difficile culture was divided into six aliquots and grown for 10 hours. During growth, EP-LR-PEG3-Biotin was added to the aliquot at sequential time points to vary the length of inhibitor incubation time. All samples were stopped simultaneously by centrifugation after 10 hours.

Figure 5. 8: Time dependency analysis of biotinylated ABP. Cultures of C. difficile 630 were incubated with EP- LR-PEG3-Biotin at 250 µM for the indicated time. Cell pellets and concentrate of culture supernatant were analysed by Western blot against LMW-SLP and NeutrAvidin-HRP.

Cell pellet Culture supernatant

EP-LR-PEG

-Biotin

α-LMW-SLP

SlpA

t(h)

NeutrAvidin-HRP blot

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The whole cell pellet and the concentrate of the culture supernatant were analysed by Western blot against LMW-SLP and NeutrAvidin to reveal the results displayed in Figure 5. 8. It was found that inhibition of S-layer processing occurred continuously and the longer the inhibitor was present in the culture, the more SlpA was found. Interestingly, the shed SlpA in the culture supernatant obeyed the same tendency as that found in the cell pellet, however unprocessed SlpA was not found in the culture supernatant until at least 4 hours of inhibitor incubation time whilst this was only 2 hours for SlpA found in the cell pellet.

Labelling of protein occurs only in the cell and not in the culture supernatant suggesting that the protease(s) involved in the cleavage remain in the S-layer. Furthermore, a similar tendency was observed as for inhibitory activity; that 2 h of inhibitor incubation time is sufficient for the labelling process to take place.

5.1.5 Strain dependence

Based on the labelling effect and inhibitory activity on SlpA processing, it was hypothesised that epoxysuccinyl inhibitors/ABPs targeted at least two proteases in C. difficile. One is involved in SlpA cleavage (inhibited by active inhibitors) and one is not (bound by inactive inhibitors and possibly also active inhibitors). It was decided to investigate this phenomenon further by looking at different C. difficile strains.

The cleavage locus of different strains still varies despite the relatively high homology around this locus, as revealed by an alignment of SlpA sequences from 35 strains from different sources (see section 1.6.2.). It is more unlikely that one specific protease can recognise all the different proteins. To address this topic, a collection of ten different strains of C. difficile which possess less homology at the cleavage site was investigated.

A panel of non-tagged and biotinylated inhibitors was chosen for this experiment. E-64 and its mimic EP-LR-NH2 and EP-LR-PEG3-Biotin were selected as positive controls, while EtEP- OH, EP-PEG3-Biotin and EP-A-PEG3-Biotin served as negative controls. EP-LGR-NH2 and EP- KTEL-NH2 were chosen to satisfy both design considerations (based on the parent compound EP-LR-NH2 and cleavage site). EP-K-PEG3-Biotin and EP-L-PEG3-Biotin were selected to mimic the P2 position of the majority of substrates (corresponding to the AA1 position of inhibitors).

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Figure 5. 9: Strain dependency of inhibitors. Amino acids indicated in red in the cleavage sequence represent the P1 position of the substrate at the cleavage site. Raw data of western blot gels are attached in the Appendix 1.

A summary is illustrated in Figure 5. 9, SlpA bands were excised and compiled in the table, while the raw data is attached in the appendix. Overall, E-64 and its mimics showed moderate activity, whereas EtEP-OH, EP-PEG3-Biotin and EP-A-PEG3-Biotin remained essentially inactive in all strains tested. Furthermore, some different levels of inhibition were observed for certain inhibitors across different strains. In particular, EP-KTEL-NH2 and EP-K-PEG3-Biotin possessed reduced or no inhibitory effects with strains including 167, E13540 and SE 538, but good activity with other strains.

As found in the reference strain 630, all biotinylated ABPs labelled a strong band at approximate 70 kDa. In some strains such as CD17, CD167 and Y, additional bands at lower molecular weights were found with lower intensity (Figure 5. 10). These bands showed similar behaviour as found with the 70 kDa bands; they were present in all samples regardless of the activity of the inhibitors. This trend might relate to a degradation process of the above mentioned proteins, but it could also result from non-specific labelling of cytoplasmic proteins released by cell lysis, as different strains might possess varied sensitivity to induce cell lysis in response to inhibitor treatment.

Results of activity and labelling indicated comparable inhibition behaviour to that found in 630. Epoxysuccinyl inhibitors may target multiple proteases, but the protease involved in SlpA cleavage might also vary between strains. Similarly to SlpA, these proteases might possess high homology among each other, but could possess subtly different active sites

Strain SlpA cleavage sequence

...P5P4P3P2 P1P1’P2’P3’... Cd 1 ...V T T K S A A K... Cd 17 ...L E T K S A D I... Cd 167 ...F S T Y R A T N... Cd 630 ...L E T K S A N D... Cd 959 ...L T T K G A T G... Cd Y ...F S S Y G K F S... SE 528 ...L D T K G A T D... R13541 ...A S T Y A S S N... R13540 ...L T T K S A T Q... R13699 ...L N T L S A S S... No Inh. E-64 EtEP- OH

EP-X-PEG3-Biotin EP-X-NH2

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appropriate for the corresponding SlpA cleavage locus. Due to the possible high homology and similarity of these proteases across all strains, a potent inhibitor could be active in most of the strains, and hypothetically a single drug could treat all strains related to CDAD if S- layer processing were to be selected as a therapeutic target. Fortunately, this ambiguity regarding the conservation of the target protease(s) was resolved in the later study described in Chapters 6 and 7.

Figure 5. 10: Labelling of targets across different strains, visualised by NeutrAvidin blot. Bacteria were grown overnight with given compounds at 100 µM. Whole cell pellet was boiled in loading buffer prior SDS-PAGE analysis.