Journal: Antimicrobial Agents and Chemotherapy

Article Title: Mutation of lipoprotein processing pathway gene lspA or inhibition of LspA activity by globomycin increases MRSA resistance to β-lactam antibiotics

doi: 10.1128/aac.01276-25

Figure Lengend Snippet: Mutation of lspA does not affect PBP2a expression levels or peptidoglycan structure and crosslinking. ( A ) Western blot of PBP2a protein in JE2, NE1757 ( lspA ), NE1757 p lspA, MSSA strain 8325-4 (negative control), and MRSA strain BHICC (positive control). Cells were grown to exponential stage in MHB 2% NaCl supplemented with 0.5 µg/mL oxacillin, with the exception of 8325-4 which was grown in MHB 2% NaCl. For each sample, 8 µg total protein was run on a 7.5% Tris-Glycine gel, transferred to a PVDF membrane and probed with anti-PBP2a (1:1,000), followed by HRP-conjugated protein G (1:2,000) and chemiluminescence detection with the Amersham ECL Western Blotting Detection kit. The blot was imaged using a LICOR C-Digit Blot scanner using the Image Studio Software (version 6.1). Four independent experiments were performed, and a representative image is shown. ( B ) Coomassie staining of the Tris-Glycine gel after transfer of protein to the PVDF membrane used in the PBP2a Western blot. ( C ) Representative UV chromatograms of peptidoglycan extracted from JE2 and NE1757 collected from cultures grown to exponential (Exp) or stationary (Stat) phase in MHB or MHB supplemented with oxacillin (OX) 3 µg/mL or 32 µg/mL. Each profile shown is a representative of three biological replicates.

Article Snippet: The blot was imaged using a LICOR C-Digit Blot scanner using the Image Studio Software (version 6.1).

Techniques: Mutagenesis, Expressing, Western Blot, Negative Control, Positive Control, Membrane, Software, Staining