3 Results

The substrate specificity of NE is poorly understood. Studies using synthetic peptides have shown that NE prefers valine at the P1 position in its substrates (Harper, et al., 1984). However, this preference cannot explain why NE cleaves virulence factors but not other proteins of Shigella, because the number of valines in the proteins of both groups is comparable. Moreover, incubation of Shigella virulence factors with purified NE (pNE) resulted in discrete cleavage products. This suggests that the Shigella proteins were folded in the Shigella culture supernatants and that NE initially has access to only a few of the potential cleavage sites.

To date, it remains unclear what NE recognizes in its substrates. To this end, we tested the following hypotheses: First, that the specificity is encoded either by the primary, secondary or tertiary structure of the substrate. Second, that a specific domain in NE determines its substrates specificity.

3.1  The Specificity of NE for virulence factors is not encoded in NE substrates

3.1.1  A recognition motif for NE was not detected in the primary sequence of IpaB

To test the first hypothesis a series of Shigella IpaB mutants were tested for their susceptibility to NE cleavage (Guichon, et al., 2001). These mutants contained individual deletions of 8-10 amino acids (aa) that spanned the coiled-coil region, as well as the putative transmembrane domains in the hydrophobic region of IpaB (figure 3.1). Coiled-coil motifs have been proposed as a common pattern for secreted virulence factors (Miao, et al., 1999;Pallen, et al., 1997) and present a possible recognition motif for NE. We reasoned that mutation in a particular NE recognition motif in IpaB should render the mutant protein resistant to NE cleavage.

To avoid false-positive results based on mis- or unfolded mutant proteins, we exclusively tested functional IpaB mutants. Since Shigella cannot invade or induce cytotoxicity in cells without a functional IpaB protein, we only tested IpaB mutants that were able to complement an ipaB deletion Shigella strain in epithelial cell invasion and macrophage cytotoxicity when introduced on a plasmid (Guichon, et al., 2001). Furthermore, these IpaB mutants were likely to be folded correctly as shown by limited proteolysis (Guichon, et al., 2001).

	Fig. 3.1: Schematic representation of IpaB mutants.
	Schematic presentation of the IpaB protein and the position of the mutations introduced (green rectangles). Adapted from (Guichon, et al., 2001).

Supernatants of S. flexneriΔipaB strains carrying the wt ipaB or the different mutant ipaB genes on a plasmid were collected and incubated with purified NE (pNE). IpaB is enriched in the supernatant because deletion of an ipa gene leads to a hypersecretory phenotype in Shigella. Although the cleavage patterns of the mutants were varying, none of the IpaB mutants was fully resistant to NE cleavage. Most mutants showed a similar cleavage pattern as the wildtype IpaB protein after treatment with pNE (figure 3.2). Interestingly, the IpaB mutant harboring the amino acid deletion Δ207-216, which is localized at the coiled-coil region, was even more susceptible to pNE cleavage than wildtype IpaB.

	Fig. 3.2: The primary sequence of IpaB does not encode for NE specificity.
	Supernatants of strains secreting wildtype or mutant IpaB protein were treated with pNE at a concentration of 100 ng/ml (+). As negative control buffer without pNE was added to each supernatant and the samples were treated equally (-). After 1 h incubation time, samples were TCA precipitated and analyzed by SDS-PAGE and immunoblotting using anti-IpaB antibody.

Taken together, this indicated that no exclusive NE recognition motif was present in these areas. Although the IpaA, B, C and D belong to different protein families, it could have been possible that NE recognizes a certain consensus sequence in these virulence factors. Therefore NE derived proteolytic products of IpaA, B and C were independently analyzed by MALDI-TOF mass spectrometry. However, such a consensus sequence was not detected (data not shown). From these experiments we concluded that the specificity of NE does not seem to be encoded in the primary sequence of NE substrates.

Next, we wanted to test whether higher order structures of the substrate are important for recognition and cleavage by NE. To this end, Shigella supernatant containing wildtype IpaB was heat-denatured and the NE cleavage assay was repeated. We observed that pNE was able to cleave native as well as denatured IpaB under the same conditions (figure 3.3; lanes 1, 2 and 6, 7). A previous study had shown that cathepsin G (CG) does not cleave Shigella virulence factors despite its high degree of homology to NE (Weinrauch, et al., 2002). Therefore we simultaneously assessed if IpaB could be rendered susceptible to CG cleavage upon denaturation. However, purified CG (pCG) neither cleaved native nor denatured IpaB (figure 3.3; lanes 3, 4 and 8, 9).

These experiments indicated that secondary or tertiary structures of the substrates are not mandatory for NE cleavage.

	Fig. 3.3: NE but not CG degrades native and denatured IpaB.
	Supernatant of Shigella (M90TΔipaB + pUC19/wt ipaB) was incubated with pNE and pCG at 100 ng/ml for 1h (lane 1-4) and 2h (lane 6-9). Aliquots of the same supernatant were heat-treated (95°C, 10 min, followed by fast cooling) prior to incubation with pNE or pCG for 1 h (lane 2, 4) and 2 h (lane 7, 9). As negative control, buffer was added to heat-treated supernatant for 2 h (lane 5). Samples were analyzed by SDS-PAGE and immunoblotting using an IpaB antibody.

3.2  The Specificity of NE for virulence factors is encoded in NE

Since we were not able to detect a recognition motif for NE in the primary or higher order structures of the NE substrate IpaB, we addressed the question of NE specificity by a structural-functional analysis of NE. We reasoned that a recognition motif for virulence proteins existed in NE and that this motif could be mutated without comprising the catalytic activity of the protease. More specifically, we hypothesized that NE mutants could exist that were still active towards its peptide substrate but would no longer recognize and cleave virulence factors like IpaB.

To this end, we selected single or multiple amino acids for mutation in NE that could present such a NE recognition motif. We based this selection on a structural comparison of NE and CG, because CG does not cleave virulence factors despite its high degree of homology to NE. We planned to compare these NE mutants to wildtype NE in their ability to degrade the NE peptide substrate and Shigella virulence proteins. For this purpose, we first had to establish an expression system that was able to yield active recombinant NE on a consistent basis. In a recent study, active recombinant NE was transiently expressed in eukaryotic rat basophilic leukemia cells (RBL-1 cells) (Li and Horwitz, 2001). However, we planned to express and purify recombinant NE in large amounts. Therefore we tried to express NE in bacteria and other non-mammalian cell systems, but we were unable to obtain active NE (see appendix 5.1). Thus, we switched to the aforementioned mammalian cell line and were finally able to express recombinant and active NE.

3.2.1  Expression of recombinant wildtype NE in mammalian cells

As mentioned above, active recombinant NE had been transiently expressed in RBL-1 cells (Li and Horwitz, 2001). The cells had been transfected with the cDNA encoding for the full-length NE protein. Since N-terminally unprocessed NE is not active, these cells likely process the full-length NE to its mature form (Li and Horwitz, 2001). Additionally, the RBL-1 cells properly targeted the enzyme to granules for storage, which is comparable to neutrophils (Gullberg, et al., 1995;Gullberg, et al., 1994).

We confirmed the transient expression of active recombinant NE using the aforementioned system (figure 3.4). The activity of NE was tested using a NE specific four amino acid peptide substrate coupled to a chromophore at the P1 position. Cleavage by NE results in the release of the chromophore leading to an increase in the optical density (OD) when measured at 410 nm wave-length. NE activity was only observed in the lysate from cells expressing NE but not in the lysate of the negative control.

	Fig. 3.4: Expression of active recombinant NE in transiently transfected cells.
	RBL-1 cells were transfected with an expression plasmid containing the cDNA encoding for the full-length NE protein (pCS2+/NE) or with an expression plasmid carrying the ß-galactosidase gene of E.coli (pCS2+/ßgal) as negative control. (A) NE activity of cell lysates from cells transfected with pCS2+/NE or with pCS2+/ßgal. The NE peptide substrate was added to the lysates and incubated for 30 min. The OD was measured at 410 nm wave-length. Lysate of non-transfected cells was treated identically and was used for normalization. All lysates were equivalent to 8x106 cells. (B) ß-galactosidase activity of the same cell lysates (1,4x104 cell equivalents). The ß-galactosidase activity was measured according to the protocol described in chapter 2.5.1.

The fact that NE could be obtained in an active form proved RBL-1 cells to be a suitable tool for NE expression. However, for the upcoming experiments it was necessary to have access to a constant reservoir of recombinant NE. Therefore a stable NE expressing cell line was generated. Since the expression vector used for the transient expression of NE did not carry an antibiotic resistance gene, the DNA encoding for the NE full-length protein was subcloned into a suitable expression vector (pcDNA3) and this construct was used to stably transfect RBL-1 cells.

The transfected plasmid can randomly integrate into the genome of the host cell. This can result in varying expression levels of a plasmid-encoded gene depending on the chromatin context of the integration site. Therefore we tested the lysates of different single-cell derived cell lines carrying the NE gene for their ability to cleave the NE specific peptide substrate (figure 3.5). Of these clones, we selected the cell line derived from clone number four because its lysate exhibited the highest NE activity. The cells did not contain endogenous NE activity since lysate from cells transfected with the empty expression vector (vector lysate) did not cleave the peptide substrate. In contrast, addition of purified NE (pNE) to vector lysate lead to high activity confirming that NE is active under the experimental

conditions. Thus, we had established stable expression of active recombinant wildtype NE, which will be designated “wt” in the upcoming experiments.

	Fig. 3.5: Expression of active recombinant wildtype NE in stably transfected RBL-1 cells.
	NE activity of lysates from 17 single-cell derived cell lines expressing NE or carrying the empty expression vector. The NE peptide substrate was added to the lysates and the OD was measured at 410 nm wave-length following a 30 min incubation period. All samples, including vector lysate that had been incubated with the substrate, were normalized against a vector lysate that had been incubated with the buffer of the substrate. As positive control, 1 μg pNE was added to vector lysate prior to addition of the substrate (V+pNE). All lysates were equivalent to 1x108 cells. Cell line number 4 is marked with an asterisk and was used for subsequent experiments.

Since whole cell lysates were used to compare the activities of wildtype and mutant NE, it was possible that the recombinant NE, although active, showed altered reactivity in this lysate background. Therefore we compared the kinetics of recombinant NE in the lysate (wt lysate) to that of purified NE (pNE).

The kinetics were tested using the same NE peptide substrate as in the previous experiments. By monitoring the OD at a wave-length of 410 nm every 30 sec over a 3 min time period, we assessed the time course of the cleavage of the substrate. The measurement started after addition of the lysate or pNE to the assay buffer containing the substrate.

We showed that wt lysate, equivalent to 4,2 x 10⁶ cells, cleaved the NE peptide substrate with linear kinetics. Additionally, its substrate turnover rate was comparable to pNE at an approximate concentration of 350 ng/ml (figure 3.6). In contrast, lysate from cells transfected with the empty expression vector (V lysate) did not degrade the peptide substrate. These data showed that the reactivity of the recombinant NE was not altered in the cell lysate.

	Fig. 3.6: Kinetics of recombinant wildtype NE.
	NE kinetics of wt and vector lysate (wt and V respectively) compared to pNE at different concentrations. All lysates were equivalent to 4,2 x 106 cells and pNE was tested at concentrations of 150, 300 and 700 ng/ml. Upon addition of the samples to the assay buffer containing the NE peptide substrate, the OD was recorded every 30 sec for 3 min at a wave-length of 410 nm.

After we had established that the reactivity of the recombinant NE towards the NE peptide substrate was comparable to that of purified NE (pNE), we wanted to test the ability of recombinant NE to cleave IpaB in Shigella supernatant. As we had shown earlier, pNE did cleave IpaB at low concentrations of 100 ng/ml (see figure 3.3).

To compare the ability of recombinant and purified NE to cleave IpaB, we used identical concentrations of active protein. Since the amount of recombinant NE in the cell lysate could vary at each cell lysis, we used an indirect method to determine the amount of active protein. First, we measured the kinetics of pNE and wt lysate within a three minute time course experiment. Because the recombinant and the purified protein both showed linear kinetics, we were able to calculate NE activity units as increase in OD per minute (OD/min). We compared the NE activity units of wt lysate to that of pNE at a defined concentration. By doing so we were finally able to deduce the amount of wt lysate that corresponded to a given NE concentration.

In the presented experiment we determined a value of 0,12 NE units for pNE at a concentration of 500 ng/ml (figure 3.7a+b). This corresponded to 0,024 units for a concentration of 100 ng/ml pNE. In contrast, wt lysate at a concentration of 500 μg total protein per ml showed an activity of 0,02 NE units (figure 3.7a+b). Therefore, we used 0,024 NE units of wt lysate and compared it to 100 ng/ml of pNE. Consequently, we added 625 μg total lysate protein to each ml of Shigella supernatant to test cleavage of IpaB by the recombinant protein.

	Fig. 3.7: Degradation of IpaB by lysates from RBL-1 cells.
	(A) NE kinetics of wt and vector lysate (wt and V respectively), of lysate from non-transfected (nt) cells, and of pNE at a concentration of 500 ng/ml. The concentration of the lysates corresponded to 500 μg/ml total protein. Upon addition of the lysates or pNE to the assay buffer containing the NE peptide substrate, the OD was recorded every 30 sec for 3 min at a wave-length of 410 nm. The data for pNE was plotted against the left y-axis whereas the data of the lysates were plotted against the right y-axis. (B) NE activity units (OD/min) of the lysates and pNE calculated as change in OD/min. The units are based on the kinetic data measured in (A). (C) Supernatant of Shigella secreting wildtype IpaB was incubated with wt, V and nt lysate at a concentration of 625 μg/ml total lysate protein and with pNE at a concentration of 100 ng/ml for 2 h. As negative control buffer without pNE was added to supernatant. Samples were TCA precipitated and aliquots were analyzed by SDS-PAGE and immunoblotting using an anti-IpaB antibody.

Unexpectedly, IpaB was not only cleaved by pNE and lysate expressing the recombinant NE but also by lysates of the negative controls (figure 3.7c). This indicated that the RBL-1 cell line itself contained an IpaB degrading activity. RBL-1 cells are basophils and thus granulocytes. Therefore they store many proteases such as tryptase, chymase or carboxypeptidase in their granules (Marone, et al., 1997). Obviously these proteases were present in the cell lysates and degraded IpaB. To be able to measure the specificity of the recombinant NE in the cell lysate, we had to inhibit the endogenous RBL proteases without affecting the activity of the recombinant NE.

It was not possible to use commercially available protease inhibitor cocktails because all of them contain general serine protease inhibitors like PMSF that also block NE. Therefore we designed an individual mixture of general and specific protease inhibitors. The general inhibitors blocked complete protease families except serine proteases, e.g. cystein- or metalloproteases, whereas the specific inhibitors suppressed, for example, chymotrypsin-like but not elastase-like serine proteases (for composition of the IC see chapter 2.5.2). First, we tested if this inhibitor cocktail (IC) affected the NE activity of the recombinant or the purified NE. Therefore we measured their kinetics in the absence and in the presence of the IC at different concentrations.

	Fig. 3.8: NE activity units of wt lysate and pNE in the presence or absence of the inhibitor cocktail (IC).
	NE activity units of buffer, wt and vector lysate (wt and V respectively), and of pNE (70 ng/ml). The lysates were equivalent to 1,7 x 106 cells. The IC (20 μM and 50 μM) and the solvent of the IC were added to the lysates or pNE and incubated at RT for 15 min. The samples were added to the assay buffer containing the NE peptide substrate and the kinetics were measured by reading the OD at 410 nm wave-length every 30 sec over 3 min. The calculated NE units are based on the kinetic data and represent the change in absorbance per minute.

We observed that the inhibitor cocktail at both concentrations did not alter the NE activity of pNE (figure 3.8). However, the NE kinetics of the wt lysate were slightly decreased at an IC concentration of 50 μM but not at 20 μM. Therefore we tested whether the IC at a concentration of 20 μM was able to block the unspecific IpaB degradation by the endogenous cell proteases. To this end, we added the IC to the cell lysates prior to incubation with the IpaB containing Shigella supernatant. Since we wanted to test the effects of the IC at maximal lysate concentrations, equal cell equivalents were added to the supernatant independent of the lysates’ activity units.

In fact, the IC at a concentration of 20 μM almost completely blocked the degradation of IpaB by endogenous RBL-1 proteases (figure 3.9, lane 4), while it did not inhibit IpaB cleavage by pNE added to vector lysate (figure 3.9, lane 5), implying that the inhibitor cocktail did not interfere with the IpaB cleaving activity of pNE. In addition, wt lysate did also cleave IpaB in the presence of the inhibitor cocktail (figure 3.9, lane 6). This proved the specificity of the recombinant wildtype NE towards the virulence factor IpaB.

Since endogenous RBL proteases were not completely inhibited by the IC at 20 μM, the concentration was increased to 50 μM in all subsequent experiments. At this concentration the inhibitor cocktail also did not interfere with the IpaB cleaving activity of purified or recombinant NE (see figure 3.16).

	Fig. 3.9: The inhibitor cocktail (IC) can block degradation of IpaB by endogenous proteases without inhibiting NE specificity.
	Supernatant of Shigella secreting wildtype IpaB was incubated with vector lysates in the presence or absence of the IC, and with wt lysate and vector lysate that contained pNE (20 ng/1x106 cell equivalent) in the presence of the IC. As controls, supernatant was also incubated with buffer (B; lane 1) and pNE in buffer (100 ng/ml) (lane 2). All lysates were equivalent to 7x106 cells. The IC (20 μM) or the solvent of the IC were added to the samples and incubated at RT for 15 min prior to addition to the supernatant. The reaction mixtures were incubated for 2 h at 37°C. After protein precipitation, aliquots were analyzed by SDS-PAGE and subsequent immunoblotting using an anti-IpaB antibody. Buffer (lane 1), pNE in buffer with solvent of IC (100 ng/ml) (lane 2), vector lysate with solvent of IC (lane 3), vector lysate with IC (lane 4), vector lysate + 140 ng/ml pNE with IC (lane 5), and wt lysate with IC (lane 6).

At this point, we had established the expression of active recombinant wildtype NE and were able to assess its specificity for the Shigella virulence factor IpaB. As a next step, we designed the NE mutants and expressed them in order to compare their activities and specificities to wildtype NE.

3.2.4  Design of NE mutants

As mentioned earlier, the proteases NE and CG share many attributes and display a striking similarity in their crystal structures. When superimposed, the α-carbons of NE and CG only display a root mean square deviation (RMSD) of 0,9 Å (see chapter 1, figure 1.8). Since a RSMD value of zero means that structures are identical in conformation (Maiorov and Crippen, 1994), it is apparent that the crystal structures of NE and CG are extremely similar (Bode, et al., 1986b;Hof, et al., 1996). Yet, NE cleaves virulence factors whereas CG does not. Thus, there had to be subtle differences that determine the opposing specificities. Therefore we examined both structures using the programs PYMOL (DeLano, 2002) and the SWISS-PDB VIEWER (Guex, 1997). Indeed, we identified single amino acids or stretches of multiple amino acids that were significantly different in NE and CG (figure 3.11). These residues were mainly located in the substrate-binding cleft formed by the ß-barrel domains of the enzymes. Interestingly, most of these amino acids were part of the previously described NE binding pockets (figure 3.10) (Bode, et al., 1989;Bode, et al., 1986b).

	Fig. 3.10 Schematic representation of the NE substrate binding pockets (SBP).
	Schematic representation of the NE substrate binding pockets (S6-S3’) that interact with the residues of a NE substrate or inhibitor (P6-P3’). The carbonyl group of the scissile bond forms hydrogen bonds with the residues of the oxyanion hole (Gly 193 and Ser 195). The residues composing the pockets are presented in the 3-letter code and their position within NE is indicated [according to chymotrypsinogen numbering (Hartley B.S., 1971)]. Some of these amino acids were mutated and tested in this study. They are highlighted in the colour corresponding to figure 3.11. Significant residues that had been suggested to influence the specificity of NE are shown in bold. This presentation is based on the crystallization of NE with the inhibitor TOM (Bode, et al., 1986b) and adapted from (Bode, et al., 1989)

Since CG does not cleave Shigella virulence factors, we assumed that replacement of the differing amino acids in NE with their structural CG counterparts might transfer the CG specificity onto the NE mutants. Consequently, NE harboring these mutations should not cleave the Shigella virulence factors any longer, but still be active towards the peptide substrate.

	Fig. 3.11: NE mutants and localization within the NE structure.
	NE was mutated by single or multiple amino acid exchanges at seven different sites. (A) Front view of the NE structure (Wei, et al., 1988) depicted as cartoon [PYMOL (DeLano, 2002)]. The residues that were mutated are shown as sticks and the different colors correspond to the different mutations indicated in (C). For orientation the C-terminus is marked (COOH). (B) Picture (A) rotated by 1800C. (C) Position of the NE residues in the sequence [numbering according to chymotrypsin (Hartley B.S., 1971)]. The residues that were exchanged or introduced are shown in the single letter code.

We exchanged four single amino acids and four amino acid stretches in NE with their structural CG counterparts. To test the influence of a non-polar and not charged residue at the positions 98, 192 and 215, we additionally exchanged these amino acids with an alanine. The mutations were introduced by site-directed mutagenesis in the NE gene encoded on the plasmid pcDNA.3/NE. The eleven NE mutants that were generated and their location within the NE structure are shown in figure 3.11.

RBL-1 cells were stably transfected with the 11 different pcDNA3/NE mutant constructs. As for the establishment of the cell line expressing recombinant wildtype NE (wt), a number of different single-cell derived cell lines for each mutant were tested for their NE activity (see appendix, chapter 5.2). Interestingly, we only observed NE activity in cell lines of seven NE mutants (35-41, N98L, F192A, F192K, A213V, F215A and F215Y). Among those only mutant F215A showed NE activity that was lower than the activity of wt NE. Of these seven mutants we selected the cell line whose lysate showed the highest NE activity per cell number for subsequent analysis.

	Fig. 3.12: NE and CG activity of different cell lines expressing NE mutant N98A
	(A) NE activity of lysates from 20 single-cell derived cell lines expressing NE mutant N98A. The NE peptide substrate was added to the lysates and the OD was measured at 410 nm wave-length after a 30 min incubation period. As positive control wt lysate was used. (B) CG activity of lysates from 6 single-cell derived cell lines expressing NE mutant N98A. Activity was measured as in (A) but the CG instead of the NE peptide substrate was used. As positive control vector lysate containing 1 μg pCG (V+pCG) was used. Clone number, marked with an asterisk, was chosen for subsequent experiments. All samples were normalized against a vector lysate that had been incubated with the respective substrate. All lysates were equivalent to 1x105 cells and treated equally.

The remaining four mutants 58A-61, N98A, 216-218 and 216-224 did not exhibit NE activity, although more than 16 single-cell derived cell lines for each mutant were tested (N98A as representative mutant is shown in figure 3.12a). This indicated that the introduced amino acids had possibly changed the specificity of the mutants for the peptide substrate. Since the introduced residues were part of the CG protein, we assumed that they had introduced a CG-like activity into these NE mutants. Therefore we assessed the ability of several cell lines of these NE mutants to cleave the CG peptide substrate. The CG peptide is identical to the NE peptide substrate except for a N-terminal modification and the P1 amino acid, which is valine in NE and phenylalanine in CG. The CG activity and kinetics were measured in an identical experimental set-up as for NE. However, RBL-1 cells contained endogenous activity against the CG peptide substrate. Therefore it was important to normalize the samples against the equal amount of vector lysate that had been incubated with the CG peptide for the identical amount of time. We observed that lysates from cells expressing NE mutants 58A-61, N98A, 216-218 and 216-224 indeed did cleave the CG peptide substrate [see appendix, chapter 5.2; (N98A as representative mutant is shown in figure 3.12b)]. Again, we selected the cell line with the highest CG activity of each mutant for further experiments.

To confirm the NE- or CG-like activities of lysates from the different mutants, we re-tested the cell lysates of all mutants for their ability to degrade the NE and the CG peptide substrate (figure 3.13). For this comparison we used the cell line of each mutant that had exhibited the highest activity towards the respective substrate. As expected, the lysates of mutants 58A-61, N98A, 216-218 and 216-224, which had cleaved the CG but not the NE substrate in the previous experiments, exclusively cleaved the CG peptide substrate. Thereby we reconfirmed the observed high activity of the lysate of mutant 216-218. In contrast, the lysates of the mutants 35-41, N98L, F192K, A213V and F215Y exclusively cleaved the NE peptide substrate. Interestingly, two mutants, F192A and F215A, cleaved both the NE and the CG substrate, albeit with different activities. Lysate from cells expressing NE F215A showed low activity towards both substrates. However, lysate from cells expressing NE F192A preferentially cleaved the NE peptide substrate. Taken together, five of the eleven mutants exclusively cleaved the NE peptide substrate, while four exclusively cleaved the CG peptide substrate. Two mutants cleaved both substrates.

	Fig. 3.13: NE and CG activity of the different NE mutants.
	NE or CG activity of lysates from cells expressing the different NE mutants. The NE (black) and CG (grey) peptide substrates were individually added to the lysates. The OD was measured at 410 nm wave-length after a 30 min incubation period. The samples were normalized against vector lysate that had been incubated with the respective substrate. As positive controls, the activities of wt lysate or of vector lysate containing 1 μg pCG (V+pCG) were measured. All lysates were equivalent to 1x105 cells and treated equally. The data represent one of five independent experiments.

We reasoned that specific inhibition of the activities of wt NE and of the different mutants would confirm that the observed substrate cleavage was caused by the recombinant proteins and not by the lysate. Therefore we assessed the abilities of the recombinant proteins to cleave the NE or CG peptide substrate in the presence or absence of specific NE and CG inhibitors. The inhibitors used in this study are both irreversible inhibitors. The NE inhibitor is identical to the peptide substrate except for the chromophore, which is replaced by a chloromethyl ketone (CMK) group. NE binds the inhibitor but its active triad cannot cleave the peptide bond of the P1 amino acid and the CMK group. Thus the enzyme inhibitor complex does not dissociate. The CG inhibitor acts in a similar way and is a three amino acid peptide with a benzyloxycarbonyl group at the N-terminus and a CMK group at the C-terminus. Like NE, CG binds this inhibitor and is incapable of cleaving the peptide bond between the P1 amino acid phenylalanine and the CMK.

	Fig. 3.14: NE activity of wt and NE-like mutants in the presence or absence of NE or CG inhibitors.
	NE activity of lysates from cells expressing wt, the NE mutants that exclusively cleaved the NE peptide substrate, or the two mutants that cleaved both substrates. The activity was measured in the absence and in the presence of (A) the NE inhibitor (NE-CMK, 200 μM (red)) or (B) the CG inhibitor (Z-GLF-CMK, 500 μM (yellow)). Inhibitor and lysates were incubated at RT for 15 min prior to addition of the NE peptide substrate. The OD was measured at 410 nm wave-length after a 30 min incubation period. Samples were normalized against vector lysate. All lysates were equivalent to 1x105 cells and treated equally. The data represent one of three independent experiments.

We observed that the NE activity of lysates from cells expressing wt or the seven NE mutants that cleaved the NE peptide substrate (35-41, N98L, F192A, F192K, A213V, F215A and F215Y) was blocked by the NE but not by the CG specific inhibitor (figure 3.14). The NE inhibitor completely suppressed the activity of the recombinant proteins independently of the different activity levels of the proteins. In contrast, the CG inhibitor did not affect the NE activities of these mutants and of wt NE, although it was used at a higher concentration than the NE inhibitor. This proved that the observed activity of wt and of the mutants was specific to the recombinant proteins.

In a second experiment, we analyzed the CG activity of the NE mutants 58A-61, N98A, F192A, F215A, 216-218 and 216-224 in the presence of the NE or CG inhibitor (figure 3.15). The CG activity of the lysates from cells expressing the mutants 58A-61 and F215A was blocked completely by the CG but not by the NE inhibitor. In contrast, the CG activity of the N98A, F192A and 216-224 lysates were affected by the CG inhibitor but not completely blocked. The NE inhibitor did only weakly interfere with the CG activity of these mutants. Unexpectedly, the CG activity of the lysate from cells expressing the mutant 216-218 was impaired to a stronger degree by the NE than by the CG inhibitor. However, since the CG activity of this lysate was extremely high, we could only observe an inhibitory effect when the lysate was diluted.

	Fig. 3.15: CG activity of CG-like mutants in the presence or absence of NE or CG inhibitors.
	CG activity of lysates from cells expressing the NE mutants 58A-61, N98A, F192A, F215A, 216-218 and 215-225. The activity was measured in the absence and in the presence of the CG inhibitor (Z-GLF-CMK, 1 mM (yellow)) or the NE inhibitor (NE-CMK, 1 mM (red)). Inhibitors and lysates were incubated at RT for 15 min before addition of the CG peptide substrate. The OD was measured at 410 nm wave-length after 30 min incubation period. Samples were normalized against vector lysate. As positive control, 1 μg pCG was added to vector lysate (V+pCG). Except for 216-218*, marked with an asterisk, all lysates were equivalent to 1x105 cells. The lysate of 216-218 was equivalent to 3,3x104 cells. All lysates were treated equally. The data represent one of three independent experiments.

In the previous experiments, we had observed that most mutants exclusively cleaved either the NE or the CG peptide substrate. Only two mutants cleaved both substrates, although one of them, F192A, preferentially cleaved the NE peptide substrate. Next, we wanted to test the ability of these NE mutants to cleave Shigella virulence factors. We assumed that the mutants that cleaved the NE peptide substrate (NE-like mutants) would act as wt NE and cleave the virulent proteins. In contrast, the four mutants that exclusively cleaved the CG peptide substrate (CG-like mutants) should behave like CG and not cleave the virulence factors. First, we assessed the ability of the NE-like mutants to cleave IpaB in the presence of the inhibitor cocktail (IC). As positive controls, we used wt lysate and pNE that had been added to vector lysate. To add equal amounts of active protein to IpaB containing Shigella supernatant, we determined the individual NE activity units of the recombinant and the purified proteins. However, we were unable to determine the kinetics of the lysates from

cells expressing the mutants A213V and F215A within the three minute time course of the experiment (data not shown). This had not been expected because these mutants had cleaved the substrate after an incubation period of 30 minutes in an earlier experiment (figure 3.13). The mutants 35-41, N98L, F192A, F192K, and F215Y as well as wt NE and pNE did cleave the NE peptide substrate with linear kinetics and we could calculate the activity units per 1,6 x10⁶ cell equivalents (figure 3.16a). We next assessed the ability of the mutants to cleave IpaB. To test different concentrations of the recombinant proteins we used mutant and wt lysates corresponding to three different activity units (0,01, 0,025 and 0,05 NE units).

	Fig. 3.16: NE mutants with NE activity cleave IpaB.
	All lysates were tested in the presence of the IC (50 μM). (A) NE activity units of lysates from cells expressing wt or the mutants 35-41, N98A, F192A, F192K, and F215Y. As second positive control next to wt, 1 μg pNE had been added to vector lysate (V+pNE; 100 ng/ 1x106 cell equivalents.). Vector lysate was used as negative control. All lysates were equivalent to 1,6 x106 cells and had been incubated with the IC (50 μM) at RT for 15 min before measurement. The NE kinetics were measured by reading the OD at 410 nm wave-length every 30 sec over 3 min. The calculated NE units represent the change in OD per minute. (B) Supernatant (1ml) of Shigella secreting wiltype IpaB was treated with the different lysates corresponding to 0,05, 0,025 and 0,01 NE units in the presence of the IC. As negative control, vector lysate was added to the supernatant. In each experiment the amount of vector lysate corresponded to the highest cell equivalence used for the other lysates. The reaction mixtures were incubated for 2 h at 370C. After protein precipitation, aliquots were analyzed by SDS-PAGE and subsequent immunoblotting using an anti-IpaB antibody.

We observed that all mutants cleaved IpaB in the presence of the IC even when low activity units were used (figure 3.16b). 0,01 NE units corresponded to a pNE concentration of 41 ng/ml, which was even lower than the concentration of 100 ng/ml used in our initial IpaB cleavage experiments (see figure 3.3). Interestingly, the IpaB cleavage pattern upon treatment with the lysate of F192K mutant implied that the activity of F192K towards IpaB was slightly reduced as compared to the other mutants. Taken together, these five mutants showed the same specificity towards IpaB like wt NE.

We also wanted to test the ability of the other six mutants to cleave IpaB. Since we could not measure the NE kinetics of the CG-like mutants and of A213V and F215A, we had to determine the amount of lysate to test for IpaB cleavage in a different way. To this end, we assessed the kinetics of wt NE, calculated its activity units (figure 3.17a) and linked this data to the concentration of total protein in the respective lysate. Since we wanted to ensure that mutants with putatively low activity could potentially cleave IpaB, we tested high activity units in this experiment. 0,05 NE units of wt lysate corresponded to a concentration of 333 μg total protein. We used the same amount of total protein of the mutant lysates and vector lysate to test for IpaB cleavage. To prove that this method of determining the lysate is reliable, we used as positive controls the lysates of the NE mutants 35-41, N98L and F215Y that had cleaved IpaB in the previous experiment. Additionally, we used vector lysate containing pNE (V+pNE) that corresponded to 0,05 NE units as positive control for wt NE.

We observed that the lysate from cells expressing the NE mutant A213V did cleave IpaB just as the positive control lysates (figure 3.17b). In contrast, F215A and the CG-like mutants 58A-61, N98A, and 216-224 did not cleave IpaB. Interestingly, incubation of IpaB with mutant 216-218 resulted in negligible IpaB degradation, which does not compare to the IpaB cleavage pattern observed upon treatment with wt or NE-like mutants.

To confirm that the CG-like mutants and F215A were active although they did not cleave IpaB like wt NE, we tested the kinetics of these mutants at similar total protein concentration using the CG peptide substrate (figure 3.17c). It is obvious that the activities of 58A-61 and F215A were low, because their activity units were hardly higher than the activity units of vector lysate. However, N98A, 216-218 and 216-224 proved to be active since they showed a higher CG activity than the vector lysate. Again, the 216-218 lysate showed an extremely high CG activity that was higher than that of 5 μg/ml pCG.

Taken together, F192A and the NE mutants that exclusively cleaved the NE peptide substrate cleaved IpaB like recombinant or purified wt NE. In contrast, NE mutants that exclusively recognized the CG peptide substrate did not, or in the case of 216-218 did only slightly target IpaB. F215A, which degraded both substrates with low activity, did also not cleave IpaB.

	Fig. 3.17: IpaB is cleaved by NE-like but not by CG-like mutants.
	All lysates were tested in the presence of the IC (50 μM). (A) NE activity units of wt lysate, vector lysate containing pNE at a concentration of 175 ng/200 μg total protein (V+pNE), and vector lysate (V). All lysates were equivalent to 200 μg total protein. The NE kinetics were measured by reading the OD at 410 nm wave-length every 30 sec over 3 min. The calculated NE units represented the change in OD per minute. For wt lysate, 200 μg total protein corresponded to 0,03 NE units. (B) Supernatant of Shigella secreting wildtype IpaB was coincubated with the different lysates at a total protein concentration of 333 μg/ml. This concentration corresponded in the wt lysate to NE activity units of 0,05 (see A). As negative control, vector lysate and, as positive control, V+pNE corresponding to 0,05 NE units were added to the supernatant. The reaction mixtures were incubated for 2 h at 37°C. After protein precipitation, aliquots were analyzed by SDS-PAGE and subsequent immunoblotting using an anti-IpaB antibody. (C) CG activity units of vector lysate and of the lysates containing the mutants 58A-61, N98A, F215A, 216-218 and 216-224. All lysates were equivalent to 100 μg total protein. As positive control, the CG kinetics of pCG at a concentration of 5 μg/ml was measured in the presence (+IC, 50 μM) and absence of the IC (no IC). The CG kinetics were measured as in (A).

It has been shown that NE cleaves membrane-bound IcsA while it does not target membrane proteins that are important for Shigella homoeostasis, e.g. the outer membrane protein A (OmpA) (Weinrauch, et al., 2002). Therefore, we tested the ability of wt and mutant NE to cleave IcsA and OmpA. We assumed that the wt NE and the NE-like mutants, including F192A, would target IcsA but not OmpA. This would prove that these recombinant proteins, just as pNE, only cleave virulence factors. To confirm that the IcsA cleavage was exclusively caused by the activity of the recombinant proteins, we tested the lysates of NE-like mutants, including F192A, and wt NE also in the presence of the specific NE inhibitor.

As in the previous experiment, we used identical total protein concentrations of the different lysates to test for IcsA and OmpA cleavage (figure 3.18a). However, in this experiment we used the total protein concentration of wt lysate corresponding to 0,01 NE units as reference because in a preliminary experiment we had observed that wt NE completely cleaved IcsA at 0,01 NE units.

We incubated Shigella with the cell lysates and analyzed the bacterial lysates for cleavage of the membrane-bound IcsA and OmpA. As expected, the wt NE and the NE-like mutants 35-41, N98L, F192A, F192K, A213V and F215Y cleaved IcsA but not OmpA (figure 3.18c). In addition, they did not cleave IcsA in the presence of the specific NE inhibitor. This proved that the recombinant proteins specifically targeted IcsA. In contrast, the mutants N98A and 216-224 did not cleave IcsA, despite being active (figure 3.18b). Interestingly, mutant 216-218 partially degraded IcsA. We speculated that this partial degradation was due to the high activity of the mutant, since 216-218 exhibited a much higher activity per total protein than the other mutants (figure 3.18c). Therefore we tested the ability of the lysate of 216-218 to cleave IcsA at a lower total protein concentration. At this concentration the activity units were comparable to the CG activity of mutant 216-224. In this experimental set-up NE mutant 216-218 did not target IcsA (figure 3.18d). Furthermore, the NE mutants 58A-61 and F215A did not show any CG activity and did not degrade IcsA.

In summary, we were able to compare the activities of recombinant wt and mutant NE towards synthetic and biological substrates. Like wt NE, the NE mutants 35-41, N98L, F192A, F192K, A213V and F215Y specifically cleaved the NE peptide substrate and the Shigella virulence factors IpaB and IcsA. In contrast, the NE mutants N98A, 216-218 and 216-224 that no longer cleaved the NE but the CG peptide substrate had lost their specificity for these virulence factors.

	Fig. 3.18: IcsA is cleaved by NE-like but not by CG-like mutants.
	All lysates were tested in the presence of the IC (50 μM). (A) NE activity units of wt lysate, vector lysate containing pNE at 75 ng/100 μg total protein (V+pNE), and vector lyste (V) in the absence or presence (+) of the NE inhibitor (NE-CMK, 200 μM). All lysates were equivalent to 100 μg total protein. The NE kinetics were measured by reading the OD at 410 nm wave-length every 30 sec over 3 min. The calculated NE units represented the change in OD per minute. For wt lysate, 100 μg total protein corresponded to 0,013 NE units. (B) CG activity units of vector lysate and of the lysates containing the mutants 58A-61, N98A, F215A, 216-218 and 216-224. All lysates were equivalent to 100 μg total protein. As positive control, the CG kinetics of vector lysate containing 5 μg pCG was measured. The kinetics were measured as in (A). (C) Shigella (M90T, 3x108/ml) was incubated with the different lysates at a total protein concentration of 52 μg/ml. For the wt lysate this concentration corresponded to 0,01 NE activity units (as described in A). As negative control, vector lysate was used. V+pNE corresponding to 0,01 NE units served as positive control. Lysates of the NE-like mutants were also tested in the presence (+) of the NE inhibitor (NE-CMK, 200 μM). The reaction mixtures were incubated for 1 h at 37 0C. After bacterial lysis, aliquots were analyzed by SDS-PAGE and subsequent immunoblotting using an anti-IcsA and OmpA antibody. (D) Shigella (M90T, 3x108/ml) was incubated with the 216-218 lysate at different concentrations of total protein. Undiluted lysate (1:1) corresponded to 52 μg/ml total protein. As negative controls, vector lysate (V) and wt lysate in the presence (+) of the NE inhibitor were used. As positive control, wt lysate in absence of the NE inhibitor was used. The experimental set-up is described in (C).