ABSTRACT
Peptidoglycan, a gigadalton polymer, functions as the scaffold for bacterial cell walls and provides cell integrity. Peptidoglycan is remodelled by a large and diverse group of peptidoglycan hydrolases, which control bacterial cell growth and division. Over the years, many studies have focused on these enzymes, but knowledge on their action within peptidoglycan mesh from a molecular basis is scarce. Here, we provide structural insights into the interaction between short peptidoglycan fragments and the entire sacculus with two evolutionarily related peptidases of the M23 family, lysostaphin and LytM. Through nuclear magnetic resonance, mass spectrometry, information-driven modelling, site-directed mutagenesis and biochemical approaches, we propose a model in which peptidoglycan cross-linking affects the activity, selectivity and specificity of these two structurally related enzymes differently.
Subject(s)
Staphylococcal Infections , Staphylococcus aureus , Humans , Peptidoglycan/chemistry , Hydrolases , Lysostaphin/analysis , Lysostaphin/chemistry , Mass Spectrometry/methods , Cell Wall/chemistryABSTRACT
Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 micrograms/ml [corrected] in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 micrograms/ml [corrected] of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.
Subject(s)
Animals, Genetically Modified , Mammary Glands, Animal/microbiology , Mastitis, Bovine/prevention & control , Staphylococcal Infections/prevention & control , Staphylococcal Infections/veterinary , Animals , Cattle , Dairying , Feasibility Studies , Female , Genetic Engineering/methods , Immunity, Innate/genetics , Lactation , Lysostaphin/administration & dosage , Lysostaphin/analysis , Lysostaphin/metabolism , Mastitis, Bovine/microbiology , Milk/chemistry , Milk/microbiology , Recombinant Proteins/administration & dosage , Recombinant Proteins/metabolism , Staphylococcus aureus/enzymologyABSTRACT
We describe a method for determination of lysostaphin activity using Remazol Brilliant Blue R (RBB)-dyed staphylococcal cells or RBB-dyed staphylococcal peptidoglycan as substrate. The dyed substrates are easy to prepare and are stable for at least 6 months. Soluble hydrolytic products released by lysostaphin are measured spectrophotometrically at 595 nm after the insoluble substrate is removed by filtration or centrifugation. The dye release assay is more sensitive and more accurate than the previously described turbidimetric assay.
