We develop of chemical tools to dissect infection mechanisms at molecular level.
Our research group focuses on molecular approaches to dissect infection mechanisms of intracellular pathogens. The research can be divided into two tracks, one dealing with posttranslational modifications as a host-pathogen crosstalk, and a second, more applied program, where we use our knowledge to combat apicomplexan parasites by interfering with their metabolisms.
Intracellular pathogenic bacteria have evolved a number of strategies to enhance both their survival and virulence during infection of a host. One such tactic is to chemically modify their eukaryotic hosts using toxin proteins that are injected via dedicated translocation machineries, like secretion systems. Once inside the host/prey cell, toxins display catalytic activity against central components of the host cell, targeting functions like cytoskeleton dynamics, vesicular trafficking and modulating the different nodes and levels of cell signaling. Most toxins employ a reactive metabolite from the host as a co-substrate. This is often a nucleotide, which is in part covalently transferred to the target proteins, thus inhibiting protein-protein interactions of the modified protein with other effectors. For example, phosphorylating toxins transfer the γ-phosphate from host cell ATP, with ADP as a leaving group, analogous to a eukaryotic kinase. On the other hand, an adenylylating (a.k.a AMPylating) toxin utilizes ATP in a different fashion. Here, the AMP moiety is attached to an amino acid residue of a host protein. In effect, the nucleobase monophosphate is transferred and pyrophosphate functions as a leaving group. This illustrates the ingenious nature of pathogens to make use of the available nucleotides.
In order to understand bacterial virulence and pathogenicity, the effect of the toxins must be considered at a molecular level. It is therefore necessary to understand the protein substrate profiles of the toxins. Although toxin targets at the protein level may appear diverse, the phenotypic outcome is surprisingly similar – on toxin exposure the typical cellular responses are that vesicular transport is redirected, cell cycle arrest is prevalent, gene transcription and cytoskeleton dynamics are attenuated and chemotaxis is inhibited. For many toxins, typically a single or, at most, a few target proteins have been identified and often in a serendipitous manner. The diverse effect that a toxin has on the host cell cannot be adequately explained by such a low number of protein targets and leads to the conclusion that: From the given orchestration of phenotype, many toxins must have more targets than those currently identified. The paucity of knowledge surrounding this field can be attributed to the fact that, to date, no generic tools exist for the specific enrichment for proteomic evaluation of the absolute toxin substrate profile.
To solve the above mentioned problems, we have developed a concept called Reactive Protein - Proteome Profiling “ RP3 ”. The goal is to create a robust method for absolute substrate profiling of bacterial toxins against cellular target proteins employing covalent nucleotide co-substrates resulting in covalent ternary complexes. No tools exist for the proteomic evaluation of the absolute substrate profile of a given toxin. Non-covalent affinity enrichment gives biased results depending on the reagent and relative target abundance. Our RP3 concept is based on covalent and subsequently cleavable capture of the target proteins by the reactive co-substrate of the toxin. This allows us – for the first time – to identify bacterial toxin substrates in an unbiased way.
In a second project, we are dealing with Cryptosporidium infection in ruminants. Cryptosporidiosis is a mammalian diarrheal disease caused by microscopic parasites of the genus Cryptosporidium, which are protozoa of the phylum Apicomplexa. Many species of Cryptosporidium exist that infect humans, as well as a wide range of commodity animals. Cryptosporidium hominis infects humans and Cryptosporidium parvum infects commodity animals and humans alike. It affects the intestines of mammals and is typically an acute short-term infection, which might become chronic in immunocompromised animals, and especially neonatal ruminants are sensitive to Cryptosporidium parvum infections, which results in life-long reduced animal health. The parasite is protected by an outer shell that allows it to survive outside the host for long periods of time and makes it highly resistant to chlorine disinfection. While the parasite might be transmitted in several different ways, water is the most common way of transmission and Cryptosporidium parvum is one of the most common causes of waterborne disease (drinking water) among humans, as well as ruminants, worldwide. Because of the minimally invasive nature of Cryptosporidium infection, innate immune responses are critical to the host's defense against infection, thus if lacking or weak, consequences are detrimental – which is the case in neonates. Cryptosporidium parasites have unique biochemistry and specific metabolic pathways compared to other Apicomplexa. Many question marks remain along the lines of development stages and infection cycle, making fundamental investigations important. This lack of knowledge originates from the absence of general transgenesis suitable for Cryptosporidium parvum, until very recently, as well as a long-term cellular in vitro propagation model.
We develop and investigate experimental drug molecules targeting essential metabolic pathways in Cryptosporidium, where no redundancy is present. This project is a collaboration between the Hedberg group and Fabrice Laurent’s laboratory at INRAE Tours in France, which allows us to investigate experimental therapies in large animal models (neonatal lambs).