Investigations on Cwp84, a Cysteine C. difficile Protease

Clostridia comprise a large family of ubiquitously occurring, anaerobic, sporulating bacteria. Whereas most clostridial species are non-pathogenic, Clostridum difficile poses a serious threat and causes most infections in clinics, leading to diarrhoea and pseudomembranous colitis. While the histotoxicity of clostridia is primarily caused by specific toxins, host infiltration and colonization are triggered by the production of various extracellular proteases such as collagen degrading metalloproteases and other proteases that recognise and degrade extracellular matrix proteins. Of particular interest is the C. difficile-specific Cwp84, a surface-bound cysteine protease conserved within all strains of C. difficile. We propose to investigate the structure and function of Cwp84 to study the molecular principles of host colonisation and infiltration by C. difficile. We discovered that the 82 kDa protease Cwp84 contains a segment with significant homology to papain-like proteases; this assignment provides a consistent explanation of the observed intermediates during auto-degradation. Together with a preliminary assignment of the Cwp84 domain architecture, we established a framework to systematically study the so far poorly understood biochemistry of the enzyme. Specifically, we will investigate the domain organisation of Cwp84 including the functional assignment of individual domains; along this line, we will reveal the activation mechanism that is related to the release of a pro- domain. Secondly, we will investigate the catalytic mechanism of the enzyme and, in particular, validate our proposed catalytic dyad (Cys116, His262) as well as the oxyanion hole (Gln110). Thirdly, we will determine the crystal structures of relevant domains of Cwp84, including the catalytic domain and complexes with active-site directed inhibitors. These structural data will be complemented by enzymological data, determined within our fourth work package. The combined data shall reveal the detailed mechanism of activation as well as important substrate recognition elements. We will confirm elements that confer Cwp84`s substrate specificity by site directed mutagenesis and engineer novel catalytic properties into the protease. This knowledge will be further exploited to design optimised active site-directed inhibitors, which represent a starting point for the development of specific diagnosis and therapy options against C. difficile infections.

Our overall aim of this research project was to clarify the biochemistry of Cwp84 protease from Clostridium difficile and to provide insights into its potential pathophysiological role especially in colonisation and/or as a virulence factor. The initial work was focused on the activity and stability modification of the cysteine protease Cwp84 from C. difficile. For this purpose a gene encoding the sequence of Clostridium difficile Cwp84 was synthetized. The substitution of the active cysteine 116 by serine (C116S) was introduced to avoid heterogeneity problems of the sample and due to the observed degradation and/or auto-activation. The dead mutant (C116S) had neither problems with poor expression nor efficient purification. The significant degradation of the Cwp84 protein observed during expression and purification process was mainly generated through a Cwp84 auto-maturation process. The cleavage sites determined by N-terminal Edman sequencing revealed that a major cleavage site at the N-terminal part of the Cwp84 occurred between K91 and S92. Surprisingly identical cleavage pattern was observed in the dead mutant C116S construct of Cwp84. As the Cwp84 protease seems to by maturate on the way of auto-activation with possible trans (intermolecular) activation elements, it could be possible, that first cleavage is done by an exogenous protease and the following one results from an auto-proteolytic mechanism. It can be concluded that the propeptide cleavage induces some conformational flexibility and simultaneously makes the surface of the catalytic domain and the active-site groove more solvent accessible. Based on our previous experience and using fluorescence-based thermal stability assays we could confirm the stabilizing effect of Ca2+ on activity modulation and stability of the protein as well as a broad pH activity range. The interactions of unprocessed Cwp84 protease with an inhibitor - cystatin C could be observed via Surface Plasmon Resonance method. This effect has an important relevance in term of protein inactivation and possible inhibition of the infection rate. However the crystal structure of the inactive Cwp84 variant is available, the exact mechanism how the bacterium acts and colonizes the gut during infection is still poorly understood. Further structural investigations are essential for complete understanding of activation/maturation processes, substrate binding and specificity of this protease. Determination of the crystal structure of full length Cwp84 in its zymogen and/or active state could provide substantial information gains regarding the full mode of action of this protein.