Probing Acanthamoeba virulence using a multi-omics approach

Free-living amoebae of the genus Acanthamoeba are highly abundant in the environment. However, accidental contact with human tissue can render these protists pathogenic, capable of causing severe diseases in humans. One common condition involves the eye where the amoebae cause a painful infection of the cornea known as Acanthamoeba keratitis (AK), most prevalent in contact lens wearers. The amoebae contaminate contact lens storage cases where they grow to high densities, and upon contact with the eye, they adhere to the cornea and progress to deeper layers. An important precondition for the infection is considered to be the cell-to-cell contact of the amoebae with the cornea cells, which activates the pathogenic cascade. However, despite advancements in recent years, our understanding of this pathogenic cascade still remains unclear. It was not until 2013 that the first genome of Acanthamoeba was published. The advent of modern omics technologies now allow for very detailed analyses of the pathomechanisms of microorganisms. In this project, we apply an integrative omics approach to investigate the contact process of Acanthamoeba to human cells in the context of AK. Our experiments are based on our long-year experience with Acanthamoeba co-culture systems and involve modern proteomics methods coupled with transcriptome sequencing. We focus on the molecules released by Acanthamoeba before, during and after contacting with cornea cells and on subsequent gene expression changes in the amoebae. The current state of knowledge indicates that a wide array of molecules, of which only very few have been identified to date, is dynamically expressed by Acanthamoeba throughout the contact process. The data generated in this project will provide novel insights into the pathogenic cascade of Acanthamoeba and thus expand the current understanding of AK pathogenesis, potentially also paving the way for novel intervention strategies.

Acanthamoeba species are free-living amoebae that are highly abundant in the environment worldwide and mainly feed on bacteria. They have a highly resilient dormant stage, the cyst, which is resistant against heat, desiccation, and most disinfectants. Thus, they can also survive in tap water, swimming pools and even contact lens containers. If they get into the eye, they can cause a painful infection of the cornea known as Acanthamoeba keratitis (AK), most prevalent in contact lens wearers. The pathogenesis of AK is not yet fully understood, however, it is generally assumed that direct cell-cell contact with cornea epithelial cells initiates a largely enzyme-based lysis of the cornea cells, the amoebae then feeding on the cell debris and multiplying in the eye. The aim of this project was to characterise and identify the molecules that are involved in the interaction of Acanthamoeba with corneal cells. To achieve this, we established Acanthamoeba co-culture systems with human corneal cells and then performed a comparative analysis of the proteins produced and secreted by the amoebae, as well as changes in gene expression within the amoebae, at various time points during cell-cell contact. We began by analysing proteolytic enzymes of Acanthamoeba, thereby identifying two novel extracellular proteases that are secreted by the amoebae immediately after contact with human corneal epithelial cells. By comparing the intracellular proteome with the respective secretome before and after contact, we were able to show that the amoebae respond to contact with human cells with a rapid activation of adhesion molecules, the production of stress proteins and the remodelling of the cytoskeleton, followed by the release of proteolytic enzymes and cytotoxic molecules. Conversely, intracellular structural proteins are largely downregulated during the initial phase of cell-cell contact. We then compared this with the interactions between Acanthamoeba and bacterial cells. Altogether, our results suggest that Acanthamoeba possess a coordinated programme for the lysis of vertebrate cells. The data collected in this project provide a detailed basis for understanding the pathogenicity of Acanthamoeba and for identifying potential target molecules for early therapeutic intervention.