Purpose: Our purpose was to identify potential surface adhesins in L. monocytogenes.
Methods: A surface protein extraction method was optimized for investigative comparison of the surface sub-proteomes and sub-transcriptomes of surface-adherent variants of L. monocytogenescells (planktonic vs. sessile) using Orbitrap LC-MS/MS and RT-qPCR, respectively. Significant protein and mRNA differential expressions were determined for multiple biological (2, 3) and technical (3, 2) tests using Fisher’s exact (P<0.02) and Student’s t-tests (P<0.05), respectively.
Results: LC-MS/MS comparative analyses for five surface extraction methods revealed that the UB-Ghost extraction method, whereby cells were pre-bled for cytoplasmic components, produced higher purity extracts (i.e. fewer number of cytoplasmic unique peptides) (153) than LiCl (190), UB (219), trypsin-BICAM-sucrose (211), and trypsin-Tris-sucrose (231), suggesting that this extraction method be used for subsequent investigations. LC-MS/MS analyses and spectral counts for surface sub-proteomes of the planktonic cells of adherence-variant strains of L. monocytogenes identified differentially expressed proteins which were primarily detected in strongly-adherent L. monocytogenes cells (≥5-fold, 43 proteins), some of which were novel moonlighting proteins. RT-qPCR gene expression analyses of 15 select surface proteins of among attached and planktonic cells of L. monocytogenes,prepared at 30°C and 42°C, revealed 7 over-expressed genes (between 2- and 102-fold expression), encoding unknown (2), virulence (3), and non-virulence (2) functions, either in sessile cells alone (3) or in both planktonic/42°C and sessile cells (4).
Significance: Food outbreaks associated with bacterial adherence may involve novel surface adhesins. Our findings suggest a group of additional surface adhesins that may contribute to a greater understanding of molecular mechanisms involved with adherence in L. monocytogenes to abiotic surfaces.