Dissecting the role of infection-driven protein mono-glycosylation in Legionella-host interaction

Humans constantly touch, swallow or inhale microorganisms such as bacteria, which exist in large numbers everywhere around us. In the vast majority of these encounters the body remains unharmed, because specialised cells of the body's defences, so called macrophages, catch the bacterial intruders, ingest and kill them. Seemingly a straight forward and simple restrictive measure, sequestration and killing of the bacteria is a highly complex process involving many proteins, which reshape the macrophage to engulf the bacteria and trigger intracellular mechanisms to dismantle them. Most bacteria do not fight back, but pathogenic bacteria employ proteins, termed effectors, to interfere with antimicrobial mechanisms. The outcome of this battle is significantly influenced by the health status of the infected person. With increasing age or underlying health conditions as well as due to poor life style choices such as smoking the capacity of macrophages to carry out this essential protective function diminishes, making it easier for pathogens to overwhelm them and cause disease.

Our understanding of the complex processes in the macrophage as well as of the weaponry of the bacteria is still superficial; however, in depth knowledge will be required to design new treatments to disarm the bacteria or boost the antimicrobial power of macrophages. Here, in this project we will use the bacterium Legionella pneumophila as model to investigate a new aspect of the interaction of bacteria and host.

L. pneumophila is usually found in fresh water bodies in the environment, but if inhaled survives and multiplies in human lung macrophages causing respiratory disease, which in the elderly or patients with compromised immunity or underlying respiratory disease can develop into Legionnaires' disease, a potentially fatal pneumonia. The exploitation of macrophages requires the Dot/Icm type IV secretion system (T4SS), a complex machinery that transports hundreds of effectors from the bacteria into the host cell, in which they manipulate processes to the benefit of the bacteria.

To carry out these manipulations some effectors modify host proteins with small chemical groups, so called post-translational modifications (PTMs). Some PTMs are used in human cells to modulate the activity of the modified protein. The effector-mediated modifications can mimic PTMs usually occurring in host cells, thus activating a natural activity of the host protein, or be new, inhibiting or reprogramming the function of the target protein.

We recently discovered that in the test tube the L. pneumophila effector LtpM modifies numerous proteins with a Glucose sugar moiety; however, how LtpM actually activates and attaches the sugar, which residues of the host targets are modified and what the role of this PTM during infection is remains unknown. Interestingly, in mammalian cells a similar, but not identical modification with one sugar moiety is an abundant PTM and used by house-keeping proteins to modulate protein activity in response to for example nutrient availability. It has been implicated in diseases such as diabetes; however, its role in the response of human cells to bacterial infection is not well characterised yet.

In this project we aim to reveal the mode of action of LtpM by determining its three dimensional structure and identifying residues, which are essential for its function. We will profile the proteins that are modified by LtpM and/or the house-keeping machinery and analyse if these two PTMs co-exist or compete. We will then determine the effect of the PTMs on the activities of the modified proteins and their role in L. pneumophila infection, promising to reveal a new bacterial warfare strategy and fundamental new knowledge about the human host response, integral information for understanding susceptibility to infection and designing new antimicrobial therapies.

In eukaryotes mono-O-GlcNAcylation, the reversible modification of proteins with one unit of O-linked b-N-acetylglucosamine, emerges as a central mechanism for translating stress and metabolic cues into altered function of proteins; however, its role in bacterial infection is unknown. Bacterial pathogens inject glucosyltransferase (GT) effector proteins into the host, where they mono-glucosylate targets. Bacterial GTs are often potent cytotoxins; however, this is not the case for the effector LtpM, a new type of GT from the respiratory pathogen Legionella pneumophila, which we discovered.

We hypothesize that under physiological infection conditions GTs like LtpM modulate protein functions mimicking or competing with mono-O-GlcNAcylation. Here, we will use LtpM and L. pneumophila as models to dissect the interplay of effector- and host-induced mono-glycosylations and establish their roles as drivers of host-pathogen interaction.

To achieve this, we will

1. Use GT sugar-donor analogues and click-chemistry to tag, purify and identify by MS the proteins in lysates from cells ectopically expressing LtpM or after addition of recombinant LtpM, which are glucosylated and/or O-GlcNAcylated and determine the modification sites.

2. Validate the targets of LtpM in L. pneumophila infected macrophages and their glucosylation and/or O-GlcNAcylation using proximity biotinylation and proteomics.

3. Characterise the catalytic mechanism of LtpM biochemically and structurally by crystallisation.

4. Determine how glucosylation and/or O-GlcNAcylation change the activity of proteins and the signalling complexes, which they form, using recombinant proteins in in vitro assays and interactome proteomics in transfected and infected cells.

5. Employ inhibitors and/or RNA interference to disable O-GlcNAcylation or deplete selected target proteins in host cells and determine the effect on intracellular survival and replication of L. pneumophila using image-based analysis.