Comparison of Listeria monocytogenes Exoproteomes from biofilm and planktonic state: Lmo2504, a protein associated with biofilms

Abstract

The food-borne pathogen Listeria monocytogenes is the causative agent of the severe human and animal disease listeriosis. The persistence of this bacterium in food processing environments is mainly attributed to its ability to form biofilms. The search for proteins associated with biofilm formation is an issue of great interest, with most studies targeting the whole bacterial proteome. Nevertheless, exoproteins constitute an important class of molecules participating in various physiological processes, such as cell signaling, pathogenesis, and matrix remodeling. The aim of this work was to quantify differences in protein abundance between exoproteomes from a biofilm and from the planktonic state. For this, two field strains previously evaluated to be good biofilm producers (3119 and J311) were used, and a procedure for the recovery of biofilm exoproteins was optimized. Proteins were resolved by two-dimensional difference gel electrophoresis and identified by electrospray ionization-tandem mass spectrometry. One of the proteins identified in higher abundance in the biofilm exoproteomes of both strains was the putative cell wall binding protein Lmo2504. A mutant strain with deletion of the gene for Lmo2504 was produced (3119Δlmo2504), and its biofilm-forming ability was compared to that of the wild type using the crystal violet and the ruthenium red assays as well as scanning electron microscopy. The results confirmed the involvement of Lmo2504 in biofilm formation, as strain 3119Δlmo2504 showed a significantly (P < 0.05) lower biofilm-forming ability than the wild type. The identification of additional exoproteins associated with biofilm formation may lead to new strategies for controlling this pathogen in food processing facilities.

Fig 1

2D-gel electrophoresis of the exoproteins of L. monocytogenes strains 3119 and J311 from biofilm cells (A and C, respectively) and from planktonic cells (B and D, respectively) on pH 3 to 7 IPG strips. Spots are numbered according to the numbering in Table 2.

Fig 2

SEM images of strain 3119 (A₁ and A₂) and strain 3119Δlmo2504 (B₁ and B₂) on stainless steel coupons. Biofilms were grown in MWB for 48 h at 25°C, followed by medium renewal and subsequent incubation for another 48 h.