Datta, S. K. | Search bioRxiv

bioRxiv2021

Loss of GdpP function in Staphylococcus aureus leads to β-lactam tolerance and enhanced evolution of β-lactam resistance

SynopsisO_ST_ABSBackgroundC_ST_ABSWe previously reported the presence of mutations in gdpP among Staphylococcus aureus strains that were obtained by serial passaging in {beta}-lactam drugs. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. ObjectivesWe sought to identify the role of gdpP in {beta}-lactam resistance of S. aureus. MethodsCDA concentrations in bacterial cytosol were measured through mass-spectrometric analysis. gdpP deletion mutagenesis and their complemented strains were created in clinically relevant S. aureus strains to characterize its function. ResultsgdpP associated mutations among passaged strains were identified to cause loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a {beta}-lactam challenge (two to three log increase in bacterial colony forming units) by promoting tolerance without enhancing MICs of {beta}-lactam antibiotics. Our results demonstrate that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level {beta}-lactam resistance and could lead to {beta}-lactam treatment failure of S. aureus infections. ConclusionsLoss of GdpP function increases tolerance to {beta}-lactams that can lead to its therapy failure and can permit {beta}-lactam resistance to occur more readily.

microbiology↗

bioRxiv2018

Prolonged culture of primary human keratinocytes isolated from suction blisters

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense is hindered by invasive isolation methods and insufficient techniques for long-term passage of primary keratinocytes in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.

cell biology↗