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.

Phonation, or our ability to speak, arises from the process of delivering a controlled exhalation of air across the vocal folds (often know as vocal cords). This causes the vocal folds to vibrate, at a frequency that can be controlled by our vocal muscles. The resultant airflow is now modulated and passes into our 'vocal tract', which is in effect our mouth. We then form the modulated air flow into sounds by muscular control of our tongue, lip and other vocal tract features. The starting point for phonation therefore is the oscillation of our vocal folds. As we exhale, the moving passage of air creates a pressure drop; this in turn causes these soft tissues to rise up, until they meet. When full closure occurs the airflow ceases, and the air pressure returns to normal, causing the focal folds to fall back again. It is this cycle, repeated at audible frequencies that creates the initial modulated airflow, that we form into sounds. This process is known as the myoelastic cycle; which was first described by Ingo Titze. Whilst visualisation of the vocal tract is readily achievable superior to the vocal folds, there is very little published data on the impact of low pressure inferior to vocal folds. Recent research has presented strong evidence that vortices form in the sub-glottal region, which will inherently aid closure by reducing the air pressure below the vocal folds. Our recently published work, using data obtained with our partners at Wisconsin Medical Centers presented data that indicated that the elastic properties of the sub-glottal mucosa were non-linear - such that their deformation under low pressure would cause a funnel effect inferior to the vocal folds. This variable deformation could offer support for the vortex theory. The study was carried out using porcine larynges. UKE Hamburg have offered us the opportunity to repeat this study using excised human donor larynges. The purpose of this OTG is to establish and enable the co-supervision of a Dotoral study that will measure and map the elastic properties of the sub-glottal mucosa in human tissue. This data will be made available to other international teams who are actively examining the importance of the aerodynamics of the vocal tract, and how it impacts on our ability to phonate. We will develop mathematical models to explain the data that is measured, and determine if it does support our theory that deformation of the mucosa below the vocal folds results in a funnel shape that promotes the creation of vortices. If our theory is correct then it indicates the importance of this region in giving us the ability to speak. In order to maximise the cost effectiveness of the grant we will also carry out trials of our innovative laser speckle skin analysis device. This has been developed to investigate if laser speckle can be used to detect lesions in skin (e.g. melanoma). We will carry out a series of experiments with excised donor human larynges to determine if laser speckle could also be deployed by phonosurgeons to detect tissue damage in vocal folds that is not visible from the surface. Other international research institutions have expressed interest in applying the laser speckle technique in new areas of research if the UKE study is successful.