Proteomic Analysis of a Hypochlorous Acid–Tolerant Listeria monocytogenes Cultural Variant Exhibiting Enhanced Biofilm Production

Abstract

Following exposure of Listeria monocytogenes Scott A (SA) to hypochlorous acid, rough colony variants were identified that were tolerant of hypochlorous acid and produced increased amounts of biofilm. A derivative of one of these variants was smooth, produced even more biofilm, and exhibited greater biofilm chlorine resistance. The objective of this research was to compare the protein expression of a cultural variant to SA and to identify proteins that might be associated with biofilm production and chlorine tolerance. Suspension chlorine tolerance for several cultural variants (SAR, SAR5, and SBS) was determined by exposure to 60 to 120 ppm of hypochlorous acid for 5 min. Hypochlorous acid tolerance of biofilms was determined after growing biofilms on stainless steel and then exposing them to 200 ppm of hypochlorous acid for 5 min. All cultural variants were able to survive 120 ppm of hypochlorous acid in suspension. There was little difference in the hypochlorous acid tolerance of the cultural variant planktonic cells. The cultural variants produced greater amounts of biofilm than the common form of L. monocytogenes and were more tolerant of hypochlorous acid. The SBS variant was selected for proteomic comparison because it was the variant that produced the most biofilm and was the most tolerant of hypochlorous acid when grown as a biofilm. Protein expression of planktonic and biofilm cells of SBS was compared to SA by two-dimensional difference gel electrophoresis. The 50s ribosomal protein, L10, was down-regulated in biofilm SBS. Other proteins down-regulated in planktonic SBS were the peroxide resistance protein (Dpr) and a sugar-binding protein (LMO0181). This sugar-binding protein was also up-regulated in biofilm SBS. One protein spot down-regulated in planktonic SBS contained both 50s ribosomal protein L7/L12 and an unknown protein (LMO1888).