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Research topic B

B1: Protein stability in Staphylococcus aureus and its impact on S. aureus pathophysiology

Investigators

Prof. Dr. Uwe Völker
Department of Functional Genomics
University Greifswald

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Publications

Dr. Ulf Gerth
Institute for Microbiology
University Greifswald

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PhD-Researcher

Larissa Busch

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Background and significance

Staphylococcus aureus can cause a wide range of diseases and is becoming a threat for human health because of the emergence of multi-resistant strains and recurrent infections. Protein homeostasis is critical for bacterial fitness and pathogenicity and thus, ATP-dependent proteases encoded by the clp genes are potential drug targets. Uncontrolled activation of ClpP by acyldepsipeptide (ADEP4) renders even dormant S. aureus and biofilms susceptible to antibiotic killing. On the other hand, inhibition of ClpP or ClpC by small molecules reduced persister formation, altered biofilm formation and reduced virulence of S. aureus. However, many of these phenomena are strongly strain dependent and anti-Clp-drugs might also have an impact on the human mitochondrial Clp-protease. We now want to define Clp-targets during conditions critical for infection as well as internalization. Promising candidates will then be selected for detailed studies in vitro and in infection settings. Furthermore, we will establish a bacterial test system for differentiating the impact of ClpP-interfering drugs on bacterial and human mitochondrial Clp-proteases.

Hypothesis and specific aims

Clp-dependent proteolysis controls temporal patterns of adaptation of S. aureus during infection mimicking stress conditions and internalization into host cells/ intracellular survival. Therefore, we will determine to which degree disturbance of this mechanism of protein homeostasis will impact fitness and virulence of S. aureus and assess if the Clp system might be a target for combatting infections.

Envisaged thesis topics

Assessment of the impact of Clp-dependent proteolysis on intracellular fitness and persistence of S. aureus during infection.


B2: Ubiquitination-dependent host cell response to Staphylococcus aureus

Investigators

Prof. Dr. med. Ulrike Seifert
Friedrich Loeffler Institute of Medical Microbiology
University Medicine Greifswald

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PhD-Researcher

Abhishek Singh

Background and significance

S. aureus induces inflammatory reactions of the host via pattern recognition receptors such as the TLRs or STING. The ubiquitin conjugation machinery reacts to inflammatory stimuli by altering post-translational modification of host cell proteins through ubiquitinating enzymes (E1, E2, E3). Ubiquitination can be reversed by the action of deubiquitinating enzymes (DUBs), proteases that cleave ubiquitin from the substrate protein or edit ubiquitin chains by the removal of single ubiquitin moieties. The type of an ubiquitin chain impacts the functional characteristics of ubiquitinated proteins by mediating protein interactions or targeting protein substrates to proteasomes, the central proteolytic unities of the UPS. Thus, the UPS is involved in the control of cell division, survival, and cell death; and it plays a crucial role in inflammation by regulating NF-κB- and inflammasome activity as well as the supply of antigenic peptides for MHC class I antigen presentation. Whereas the role of ubiquitination has been extensively studied in infections with viruses or Gram-negative bacteria, the function of ubiquitin modification in host cells during Gram-positive bacterial infection is largely unknown.

Envisaged thesis topic

Structural and functional analysis of the ubiquitin-proteasome system in cells infected with S. aureus.


B3: Elucidating structure and function of CE-clan protease-related bacterial virulence factors with dual ubiquitin-protease and acetyltransferase activity

Investigators

Prof. Dr. rer. nat. Michael Lammers
Institute of Biochemistry
University Greifswald

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Publications

Prof. Dr. rer. nat. Elke Krüger
Institute of Medical Biochemistry and Molecular Biology
University Medicine Greifswald

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PhD-Researcher

Ole Schmöcker

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Background and significance

CE-clan protease-related proteins are injected into their host cells by various intracellular Gram-negative bacteria. Some members of the CE-clan are proteolytic deubiquinating enzymes (DUBs), others have acetyltransferase activity (AcT), and still others can perform both enzymatic functions with the same catalytic center. However, the enzymatic activities of many CE-clan protease-related factors, their cellular substrates and their interaction partners are unknown. We will investigate how the DUB/AcT functions of bacterial CE-clan proteases manipulate the ubiquitin-modification system of the host cell to their advantage during infection. CE-clan proteases are an ideal model system for elucidating structure-function relationships in proteases. Their study will provide insights into protease evolution. Moreover, as a long term goal these studies might unravel novel therapeutic strategies to fight bacterial infections.

Hypothesis and specific aims

Our aim is to understand how bacterial CE-clan enzymes use their DUB and/or AcT activity to manipulate the host cell for an efficient infection process. Functional and structural studies on selected DUB/AcT candidates will reveal the enzymatic activities and the underlying catalytic mechanism to unravel molecular determinants for DUB activity and chain-type specificity and/or AcT activity and will allow to derive information on the evolution of these CE-clan related enzymes. Cellular studies and mass-spectrometry will reveal cellular processes targeted by the DUB and/or AcT activity. We define the following aims:

Aim 1. To functionally characterize selected CE-clan enzymes.

Aim 2. To structurally define determinants for DUB and/or AcT activity.

Aim 3. To unravel the physiological impact of DUB/AcTs in human cells.

Aim 4. To identify novel substrates/interaction partners for selected DUB/AcTs.

Methods to test hypotheses

To understand how CE-clan related proteases of pathogenic Gram-negative bacterial pathogens applies different catalytic strategies to manipulate cellular processes in the host cells that allow an efficient infection process, we will apply various techniques:

A) Recombinant protein production using different expression systems (Escherichia coli and/or Saccharomyces cerevisiae).

B) Purification of proteins using diverse strategies applying automated FPLC-systems.

C) Biochemical characterizeation of enzymes and proteins (Western blotting, enzyme kinetics, DUB/AcT assays, etc.).

D) Biophysical characterization of protein structure and function using diverse techniques such as isothermal titration calorimetry (ITC), fluorescence spectroscopy and X-ray crystallography.

E) Cell biology and mass spectrometry to understand the cellular processes that are targeted by CE-clan related virulence factors, including confocal fluorescence microscopy, and to identify cellular targets.

Envisaged thesis topic

Elucidating structure and function of CE-clan protease-related bacterial pathogenicity factors with dual ubiquitin-protease and acetyltransferase activity


B4: Altered immune responses upon proteasome impairment

Investigators

Prof. Dr. rer. nat. Elke Krüger
Institute of Medical Biochemistry and Molecular Biology
University Medicine Greifswald

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PhD-Researcher

Sophie Möller

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Background and significance

During immune responses, protein homeostasis (proteostasis) is challenged by synthesis of pathogen proteins as well as by inflammatory damage conferring severe proteotoxic stress. Clearance of damaged proteins as well as precision proteolysis of signaling factors by the ubiquitin proteasome system (UPS) play a pivotal role in immune cells. Dysregulation of this fine-tuning promotes metabolic changes, induce cell death, or alters the production of cytokines and other immune regulatory components. The unfolded protein and integrated stress responses integrate cellular signaling upon proteotoxic stress in order to decide cell fate between repair and death (10.3389/fimmu.2019.02756 ).

The important function of the proteasome in the control of immune responses becomes evident by proteasome dysfunction in patients with inborn errors of the proteasome, who suffer from systemic autoinflammation with interferonopathy.

The main goal of this research project is to elucidate the mechanisms, by which proteasome impairment alters immune cell function. In the project we will use patient’s white blood cells or mouse models and apply immunological, biochemical, or molecular and cellular biology techniques.

Envisaged thesis topic

Altered signaling upon proteasome impairment and its impact on immune cell function


B5: Role of UPS-dependent protein degradation in S. pneumoniae- and S. aureus-induced heart failure

Investigator

Prof. Dr. med. Jens Fielitz
Dept. of Internal Medicine B
University Medicine Greifswald

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PhD-Researcher

Celina Würner

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Background and significance

Sepsis is the leading cause of death in critically ill patients; and septic cardiomyopathy contributes to this outcome. Septic cardiomyopathy occurs in half of septic patients leading to a mortality of up to 70%. The mortality of septic patients is directly associated with both systolic  and diastolic  cardiac dysfunction. Infections causing inflammation and sepsis are major risk factors for heart failure. For example, 20% of adults hospitalized for pneumococcal pneumonia experience congestive heart failure, arrhythmia, and myocardial infarction. The pathogenesis of septic cardiomyopathy is not well understood, but functional as well as structural abnormalities of the heart are implicated. Likewise, the complex interplay between the infection with specific bacteria, the accompanying inflammation and their effects on cardiac function are not well defined.

Hypothesis and specific aims

MuRF1-mediated and UPS-dependent protein degradation causes heart failure in S. pneumoniae- and S. aureus-infection.

Aim 1. To investigate molecular targets and pathways mediating S. pneumoniae- and S. aureus infection-induced heart failure

Aim 2. To understand how S. pneumoniae- and S. aureus infection activate protein degradation in cardiomyocytes

Envisaged thesis topic

Mechanisms of S. pneumoniae- and S. aureus-induced cardiomyocyte dysfunction and heart failure


B6: Roles of phagocyte proteases in defense against Mycobacterium tuberculosis

Investigators

Prof. Dr. Anca Dorhoi
Institute of Immunology
Friedrich-Loeffler-Institut
Federal Research Institute for Animal Health

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PhD-Researcher

Faisal Shah

Background and significance

Host proteases enriched in myeloid cells control generation of inflammatory mediators and remodel tissue during infection. In phagocytes, Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), accesses compartments with distinct proteolytic capacities. Experimental evidence supports a host-protective role of cysteine (cathepsin B, S and L) and serine (cathepsin G) proteases in macrophages. These proteases are present in endosomes in macrophages and also in the neutrophils’ granules. The TB granuloma, as well as foci of TB inflammation, are hypoxic and proteolysis in phagocytes under hypoxic environment remains unknown.

Hypothesis and specific aims

Intracellular proteolysis may alter the release of cytokines and cell activation and could be affected by low oxygen tension.

Envisaged thesis topic

The project will evaluate proteolysis in Mtb-infected phagocytes under hypoxic conditions and its impact on inflammation.


B7: Endogenous inhibition of cathepsin B regulates intracellular protease activation during acute pancreatitis

Investigators

PD Dr. Ali Aghdassi
Dept. of Internal Medicine A
University Medicine Greifswald

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Dr. rer. nat. Matthias Sendler
Dept. of Internal Medicine A
University Medicine Greifswald

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PhD-Researcher

Saeedeh Asgarbeik

Background and significance

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 intracellular secretory compartments. Recent data suggest that not only the activation of trypsinogen is crucial for cell death, but that also CTSB plays an important role in the induction of apoptosis and necrosis. Even pyroptotic cell death of infiltrating macrophages depends highly on cytosolic redistribution of CTSB, leading to inflammasome activation, cytokine secretion and systemic hyperinflammation. To counteract uncontrolled CTSB activity, acinar cells express various cysteine protease inhibitors. In addition to the inhibition of CTSB, they also control the activity of cathepsin L (CTSL), another lysosomal protease, which has a protective effect in pancreatitis by degrading active trypsin. CTSL also plays a central role for the regulation of apoptotic cell death and represents the antagonist of CTSB in this cascade. The regulation of the CTSB and CTSL by activation and inhibition is not only crucial for the activation of trypsinogen, but also the induction of cell death depends on the activity of CTSB.

Hypothesis and specific aims

We assume that the intracellular activity of cathepsin B is regulated by the presence of active endogenous inhibitors. The inhibition of cathepsin B is crucial for the regulation of protease activation, cell death and determines the apoptosis/necrosis balance.

Aim 1. To explain the roles of endogenous protease inhibitors during pancreatitis.

Aim 2. To elucidate the effects of cytosolic endogenous inhibitors.

Envisaged thesis topic

Protease inhibitors regulate the proteolytic activation of trypsinogen during acute pancreatitis and determine the cell death signalling in pancreatic acinar cells.