human skin
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Staphylococcus aureus metabolic requirements during skin colonization
Project Summary Staphylococcus aureus causes 76% of all skin infections, and yet simultaneously this pathogen asymptomatically colonizes the skin of 8-22% of healthy adults. Since the majority of S. aureus disease is the result of autoinfection from the colonizing strain, and invasive infections often originate from the skin, there is an urgent need to understand colonization mechanisms. In colonizing the skin, S. aureus encounters abundant levels of amino acid derivatives like urocanic acid and 5-oxoproline (OP) that contribute to the skin’s “acid mantle” and have reported anti-Staphylococcal properties. The central hypothesis of this project is that amino acid transport and catabolism is a critical feature of S. aureus skin colonization. To model this environment, we developed a skin-like media (SLM) to assess S. aureus physiology on the human skin surface. We determined the S. aureus transcriptional response using RNAseq and performed metabolomics in SLM, both of which demonstrated that amino acid catabolism genes are upregulated and that amino acids are rapidly consumed. These findings indicate that S. aureus has a skin expression program that enables survival and growth in this harsh environment. In Specific Aim 1, we are investigating S. aureus metabolism of serine, the second most abundant amino acid on human skin. We hypothesize that serine transport and catabolism is critical for S. aureus skin colonization. We will assess growth of mutant strains disrupted in serine pathways in the SLM and during mouse skin colonization. With 13C-tracing experiments we will investigate serine flux in S. aureus using metabolomics. We will determine serine transport mechanisms using bioinformatic guided targets and serine analogues. In Specific Aim 2, we will assess S. aureus resistance to toxic skin metabolites. OP is abundant on human skin and is known to be deleterious to bacteria. Our preliminary metabolomics studies indicate that S. aureus metabolizes OP in SLM, and we have identified a putative oxoprolinase (genes SAUSA300_1566-1561) that is upregulated on skin. We hypothesize that the detoxification of OP contributes to S. aureus survival on the skin. We will construct mutants in the 1566-1561 locus and test their contributions to OP metabolism in SLM with growth and metabolomics experiments. We will also investigate OP transport and test mutant strains in our mouse skin colonization model. In Specific Aim 3, we will identify new determinants of S. aureus skin colonization using TnSeq. We have developed an improved TnSeq library preparation and analysis protocol, and in our preliminary studies we performed TnSeq in SLM and in our mouse skin colonization model. We will evaluate pathway hits, such as respiration and fermentation, and aspartate metabolism targets by testing constructed mutants during SLM growth and in the mouse model. Novel hits will be validated with follow-up genetic experiments and 13C-tracing experiments. Collectively, the proposed studies will advance our knowledge of S. aureus colonization and adaptation to the skin environment.
Microneurography And Microstimulation Of Single Tactile Afferents In The Human Hand
Microneurography is a method, invented by Ake Vallbo and Karl-Erik Hagbarth in the late 1960, with which we can record the activity from single, identified nerve fibres in awake human participants. In this talk, I will then discuss the method, its advantages and limitations, and some of the key discoveries regarding coding of tactile events in the signalling from receptors in the human skin. An extension of the method is to stimulate single afferents, and record the resulting tactile sensations reported by the participants, so-called microstimulation. The first experiments were done in the 1980s, but the method has recently seen a revival, and is currently being combined with high-resolution brain imaging in the study of the relationship between tactile nerve signals, sensations, and processing of tactile information in the brain.
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