Research topic C

Proteases play central roles in host pathogen interactions. On the pathogen side they enable adaptation to the host and pathogenesis, on the host side they are involved in immune defense mechanisms. The elucidation of the function of particular bacterial and host proteases in the infection process requires the identification of their substrates and the characterization of the functional properties of these proteases. Proteomics approaches such as the comparative assessment of protein synthesis rates and stabilities in wild type cells and mutants lacking individual proteases will enable an unbiased identification of such substrates. Here, we will employ these technologies to analyze proteases from S. aureus and phagocytes.

The ability of S. aureus to thrive in different host environments is facilitated by host-specific virulence factors, including proteases. Both the inactivation of superfluous proteases by nonsense mutations and the acquisition of novel protease genes have been described for S. aureus lineages adapted to non-human hosts. Staphylococcal proteases can mediate multiple functions and contribute to S. aureus pathogenicity by destroying host extracellular matrix and by evading or manipulating host immune responses.

 Neutrophils are rich in proteases, which can be exocytosed upon degranulation during infection. Tuberculosis (TB) patients present high systemic levels of neutrophil granular proteins. TB lesions, notably necrotic granulomas and cavities, contain large numbers of neutrophils and are sites of bacillary replication and transmission. The low oxygen levels, which characterize these lesions, increment neutrophil degranulation. Extracellular proteolysis as a consequence of neutrophil activation may alter features of Mycobacterium tuberculosis with relevance for disease outcome.

(1) The protease Jep mediates host specificity by targeting murine host proteins.

(2) Enhanced proteolysis occurs in hypoxic lesions and extracellular proteolysis subsequent to neutrophil degranulation alters the Mtb lipo-proteome.

1. Functional analysis of the novel Staphylococcus aureus serine protease Jep.
2. Function of neutrophil proteases in Mtb virulence – Profiling of substrates in the lipoproteome of Mtb.

S. aureus serine protease-like proteins (Spls) elicit a type 2- biased immune response in susceptible individuals. In one case (SplB) cleavage of the IL-1 family member IL-33 has been demonstrated. These Spls are released in high amounts by the majority of S. aureus clinical isolates. Some of their cleavage sites have been identified, but do not permit prediction. In particular their preferred (patho)physiological substrates are largely unknown. Furthermore, S. pneumoniae serine proteases are thought to contribute to barrier disruption and immuno­modulation. This project therefore aims to clarify these important aspects using advanced methods of enzyme engineering to provide a detailed understanding of the sequence-structure-function relationships of the SpIs and S.pneumoniae serine proteases and  their immune response.

We will use a range of protein engineering methods, including ultra-high-throughput screening tools to reach the project aims. This will include phage display and DNA shuffling to identify mutations that modulate the SpI or S. pneumoniae serine protease substrate specificity.

Aim 1. To identify specific cleavages sites of SpIs or S.p. serine proteases substrate specificities to relate this to their function.

Aim 2. To understand the impact of sequence differences between S.p. serine proteases on substrate specificities.

Sequence-structure-function analysis of Streptococcus and Staphylococcus extracellular serine proteases

Acute pancreatitis is characterized by self-digestion of the pancreas by its own proteases. The premature and intracellular activation of digestive enzymes in exocrine cells of the pancreas triggers the process and leads to cell death. The first step is the cleavage of trypsinogen to trypsin by the lysosomal hydrolase cathepsin B (CTSB), which is present in the same intra-cellular secretory compartments. However, many details about this protease as well as other proteases are not clear. This project will hence apply a broad range of enzymology techniques as well as perform protein engineering studies to elucidate the distinct biochemical functions of proteases centrally involved in acute pancreatitis.

We argue that in homeostasis, premature trypsin formation by CTSB is primarily prevented by an inhibitor protein. However, if this inhibitor protein is cleaved by trypsin, a fragment with the opposite function is formed: It stabilizes CTSB activity leading to increased trypsin formation in the sense of a vicious circle. This process is further accelerated by proteolytic inactivation of a distinct trypsin inhibitor by CTSB.

Aim 1. To elucidate the function of relevant proteases involved in acute pancreatitis.

Aim 2. To clarify the effects of other endogenous inhibitors.

Sequence-structure-function analysis of proteases involved in acute pancreatitis