Crosstalk of Keratinocytes with Commensals and Neutrophils shapes Staphylococcus aureus Skin Colonization

Our skin is constantly exposed to a large number of pathogens while at the same time undergoing selective colonization by commensal microorganisms such as Coagulase-negative Staphylococci. Staphylococcus aureus, however, is a facultative pathogen that is usually absent from healthy skin but frequently colonizes the inflamed skin of atopic dermatitis (AD) patients where it further promotes inflammation. Thereby, increasing S. aureus colonization was shown to correlate with a loss of microbiome diversity indicating a role for skin commensals to shape pathogen colonization. Keratinocytes, as the most abundant and outermost cell type in the epidermis, need to discriminate commensals from pathogens and orchestrate subsequent immune reactions in response to colonizing microbes. However, the mechanisms how individual commensals cooperate with keratinocytes and the immune system of the skin to prevent pathogen colonization are barely understood. Therefore, this work aimed at investigating the functional effects of two skin commensals, S. epidermidis and S. lugdunensis, on S. aureus skin colonization. Using an in vitro adhesion and invasion assay with primary human keratinocytes and an epicutaneous mouse skin colonization model we show that pretreatment with S. epidermidis or its secreted factors significantly reduces S. aureus skin colonization. However, in this work we also demonstrate that this protection is dependent on the integrity of the epithelial barrier and is completely lost during skin inflammation. Using the same models, we further demonstrate that the S. lugdunensis-derived cyclic peptide antibiotic, lugdunin, which was previously shown to inhibit S. aureus epithelial colonization, induces a similar protective effect in human keratinocytes and mouse skin. Additionally, lugdunin can amplify the S. epidermidis-induced effect. Further analysis revealed that, beyond its bactericidal activity, lugdunin also possesses TLR/MyD88-dependent immune-modulatory activities which lead to expression and release of LL-37 and CXCL8/MIP-2 in human keratinocytes and mouse skin as well as to the recruitment of monocytes and neutrophils in mouse skin. Ultimately, synergistic antimicrobial activity in combination with skin-derived AMPs indicates that lugdunin is a multi-functional peptide providing host protection against S. aureus by multiple mechanisms. Since S. aureus frequently colonizes the inflamed skin of AD patients, this work also aimed at understanding how inflammation contributes to the initial skin colonization event with S. aureus. We found that recruited neutrophils in response to tape-stripping enhance S. aureus colonization. These findings were confirmed using an in vitro co-culture model with keratinocytes and neutrophils. Further analysis of the mechanism demonstrated that neutrophil extracellular traps (NETs) influence keratinocytes in a way that favors S. aureus colonization. Finally, we show that S. epidermidis can reduce neutrophil recruitment induced by S. aureus, which might partly explain how microbiota contribute to pathogen skin protection during homeostasis. At the same time this work suggests that during skin inflammation the release of NETs by infiltrating neutrophils superimposes the microbiota-mediated skin protection and might thus result in enhanced S. aureus colonization. In conclusion, this work describes how two members of our skin microbiota, S. epidermidis and S. lugdunensis, can prevent S. aureus skin colonization. Thus, it delineates new ways how commensals protect our skin from pathogens. In addition, the work presented here also illustrates that S. epidermidis contributes to increased S. aureus colonization during skin inflammation and that the recruitment of immune cells can have conflicting effects. Consequently, the signals of the skin microbiome need to be interpreted in the corresponding microenvironment

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein-like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat-inducible and appears to play a major role in Lpl1 interaction. Pre-incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1-Hsp90 interaction induces F-actin formation, thus, triggering an endocytosis-like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl-Hsp90 interaction.Fil: Tribelli, Paula Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Eberhard Karls Universität Tübingen.; Alemania. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Luqman, Arif. Eberhard Karls Universität Tübingen.; AlemaniaFil: Nguyen, Minh Thu. Eberhard Karls Universität Tübingen.; AlemaniaFil: Madlung, Johannes. Eberhard Karls Universität Tübingen.; AlemaniaFil: Fan, Sook Ha. Eberhard Karls Universität Tübingen.; AlemaniaFil: Macek, Boris. Eberhard Karls Universität Tübingen.; AlemaniaFil: Sass, Peter. Eberhard Karls Universität Tübingen.; AlemaniaFil: Bitschar, Katharina. Eberhard Karls Universität Tübingen.; AlemaniaFil: Schittek, Birgit. Eberhard Karls Universität Tübingen.; AlemaniaFil: Kretschmer, Dorothee. Eberhard Karls Universität Tübingen.; AlemaniaFil: Götz, Friedrich. Eberhard Karls Universität Tübingen.; Alemani

Ubiquitin orchestrates proteasome dynamics between proliferation and quiescence in yeast.

Proteasomes are essential for protein degradation in proliferating cells. Little is known about proteasome functions in quiescent cells. In nondividing yeast, a eukaryotic model of quiescence, proteasomes are depleted from the nucleus and accumulate in motile cytosolic granules termed proteasome storage granules (PSGs). PSGs enhance resistance to genotoxic stress and confer fitness during aging. Upon exit from quiescence PSGs dissolve, and proteasomes are rapidly delivered into the nucleus. To identify key players in PSG organization, we performed high-throughput imaging of green fluorescent protein (GFP)-labeled proteasomes in the yeast null-mutant collection. Mutants with reduced levels of ubiquitin are impaired in PSG formation. Colocalization studies of PSGs with proteins of the yeast GFP collection, mass spectrometry, and direct stochastic optical reconstitution microscopy of cross-linked PSGs revealed that PSGs are densely packed with proteasomes and contain ubiquitin but no polyubiquitin chains. Our results provide insight into proteasome dynamics between proliferating and quiescent yeast in response to cellular requirements for ubiquitin-dependent degradation