SummaryStaphylococcus aureus, which is a leading cause of hospital-acquired infections, binds via fibronectin to integrin 51, a process that can promote host colonization in vivo. Integrin engagement induces actin cytoskeleton rearrangements that result in the uptake of S. aureus by non-professional phagocytic cells. Interestingly, we found that fibronectin-binding S. aureus trigger the redistribution of membrane microdomain components. In particular, ganglioside GM1 and GPI-linked proteins were recruited upon integrin 1 engagement, and disruption of membrane microdomains blocked bacterial internalization. Several membrane-microdomain-associated proteins, such as flotillin-1 and flotillin-2, as well as caveolin, were recruited to sites of bacterial attachment. Whereas dominantnegative versions of flotillin-2 did not affect bacterial attachment or internalization, cells deficient for caveolin-1 (Cav1 -/-) showed increased uptake of S. aureus and other Fn-binding pathogens. Recruitment of membrane microdomains to cell-associated bacteria was unaltered in Cav1 -/-cells. However, fluorescence recovery after photobleaching (FRAP) revealed an enhanced mobility of membranemicrodomain-associated proteins in the absence of caveolin-1. Enhanced membrane microdomain mobility and increased uptake of S. aureus was repressed by expression of wild-type caveolin-1, but not caveolin-1 G83S, which harbors a point mutation in the caveolin scaffolding domain. Similarly, chemical or physical stimulation of membrane fluidity led to increased uptake of S. aureus. These results highlight a crucial role for caveolin-1 in negative regulation of membrane microdomain mobility, thereby affecting endocytosis of bacteria-engaged integrins. This process might not only limit host cell invasion by integrin-binding bacterial pathogens, but might also be physiologically relevant for integrin-mediated cell adhesion.

We report here the cloning and characterization of a cytoplasmic kinase of Clostridium acetobutylicum, named MurK (for murein sugar kinase). The enzyme has a unique specificity for both amino sugars of the bacterial cell wall, N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), which are phosphorylated at the 6-hydroxyl group. Kinetic analyses revealed K m values of 190 and 127 M for MurNAc and GlcNAc, respectively, and a k cat value (65.0 s ؊1 ) that was 1.5-fold higher for the latter substrate. Neither the non-Nacetylated forms of the cell wall sugars, i.e., glucosamine and/or muramic acid, nor epimeric hexoses or 1,6-anhydro-MurNAc were substrates for the enzyme. MurK displays low overall amino acid sequence identity (24%) with human GlcNAc kinase and is the first characterized bacterial representative of the BcrAD/BadFGlike ATPase family. We propose a role of MurK in the recovery of muropeptides during cell wall rescue in C. acetobutylicum. The kinase was applied for high-sensitive detection of the amino sugars in cell wall preparations by radioactive phosphorylation.Clostridia are anaerobic, spore-forming, and A/T-rich Gram-positive rods that include several toxin-producing pathogens (e.g., Clostridium difficile, C. botulinum, C. tetani, and C. perfringens), as well as nonpathogenic members such as C. acetobutylicum ATCC 824. The latter is a biotechnologically important strain that is used for the production of solvents such as acetone, butanol, and ethanol by fermentation of a broad range of mono-, di-, and polysaccharides (13). Although solvent formation by the conversion of carbohydrates attracted interest in the physiology of carbohydrates in this organism (17) and despite its wide use in metabolic engineering and the recent sequencing of its genome (20), the cell wall (peptidoglycan) catabolism has not been investigated thoroughly in C. acetobutylicum or in any of its pathogenic relatives. For the related members of the firmicutes, Bacillus subtilis, B. megaterium, and B. cereus, it was shown that as much as 50% of the parental peptidoglycan is turned over in one generation (6,16,21). We have recently identified a pathway in B. subtilis for the rescue of peptidoglycan fragments, which involves the sequential processing of N-acetylglucosamine (GlcNAc)-N-acetylmuramic acid (MurNAc) peptides (muropeptides) by the secreted B. subtilis enzymes N-acetylglucosaminidase (NagZ Bs ) and Nacetylmuramyl-L-alanine amidase (AmiE Bs ) (14). Thereby, the amino sugars GlcNAc and MurNAc are generated extracellularly and are presumably further metabolized alike in Escherichia coli. The pathways involve the uptake by specific phosphotransferase system transporters, NagP (25) and MurP (8), respectively, yielding, intracellularly, the respective 6-phosphate sugars, the cytoplasmic recycling enzyme MurNAc-6-phosphate etherase (MurQ), which converts MurNAc-6-phosphate to GlcNAc-6-phosphate (10, 14), and the nag genes (nagA-encoded GlcNAc-6-phosphate deacetylase and nagBencoded glucosamine-6-phosphate deaminase), w...

Due to the rise in multidrug resistance, Staphylococcus aureus, a Gram-positive coccoid bacterium, is becoming a major healthcare challenge on a global scale (Liu et al., 2011;Sieradzki et al., 1999;Tsiodras et al., 2001;Turner et al., 2019). This microbial pathogen is able to cause a spectrum of diseases, from chronic manifestations such as abscesses, chronic osteomyelitis, or mastitis to lifethreatening acute diseases such as endocarditis, sepsis, or toxic shock syndrome. S. aureus produces numerous bacterial surfaceassociated and secreted virulence factors, which allow the pathogen to adhere to and invade otherwise nonphagocytic cells, to evade immune detection, or to intoxicate host tissues (Arciola et al., 2018;Foster et al., 2014). Among staphylococcal adhesins, the cell wallanchored fibronectin-binding proteins (FnBPs) play a particularly prominent role in host cell invasion (Foster, 2016;Hauck et al., 2012;Sinha et al., 1999). FnBPs can capture the glycoprotein fibronectin (Fn), which is an abundant constituent of human blood plasma

The "Rapid Ag test based on Alpaca Nanobodies" is a test developed with single domain antibodies. This type of antibody is capable of effectively recognize the Spike protein on the surface of SARS-CoV2 leading a signal that is 10 times stronger than conventional antibodies. It takes only 16 minutes from sample to result. The procedures for using this test were designed to ensure an easy use and interpretation, making it a quick and simple test.