Comparison of Surface Proteomes of Adherence Variants of Listeria Monocytogenes Using LC-MS/MS for Identification of Potential Surface Adhesins

Abstract

The ability of Listeria monocytogenes to adhere and form biofilms leads to persistence in food processing plants and food-associated listeriosis. The role of specific surface proteins as adhesins to attach Listeria cells to various contact surfaces has not been well characterized to date. In prior research comparing different methods for surface protein extraction, the Ghost urea method revealed cleaner protein content as verified by the least cytoplasmic protein detected in surface extracts using LC-MS/MS. The same technique was utilized to extract and detect surface proteins among two surface-adherent phenotypic strains of L. monocytogenes (i.e., strongly and weakly adherent). Of 640 total proteins detected among planktonic and sessile cells, 21 protein members were exclusively detected in the sessile cells. Relative LC-MS/MS detection and quantification of surface-extracted proteins from the planktonic weakly adherent (CW35) and strongly adherent strains (99-38) were examined by protein mass normalization of proteins. We found that L. monocytogenes 99-38 exhibited a total of 22 surface proteins that were over-expressed: 11 proteins were detected in surface extracts of both sessile and planktonic 99-38 that were ≥5-fold over-expressed while another 11 proteins were detected only in planktonic 99-38 cells that were ≥10-fold over-expressed. Our results suggest that these protein members are worthy of further investigation for their involvement as surface adhesins.

RT-qPCR Analysis of 15 Genes Encoding Putative Surface Proteins Involved in Adherence of Listeria monocytogenes

“…The expression profiles of these genes (i.e., lmo0202, lmo0434, lmo1293, lmo2505, lmo2656, lmo2713) were consistent with the protein profiles attained with LC-MS/MS for surface extracts of the 99-38 Listeria cells attached to beads (Table 3A) [24]. Of four abundant proteins detected by LC-MS/MS in surface extracts from planktonic cells of 99-38 at 30 °C [24], only one member (lmo0723) correlated with gene expression profiles in this study. These inconsistent profiles could be partly due to the competitive (physical) detection of protein abundancy by LC-MS/MS vs. targeted gene expression studies using real-time RT-PCR as explained by others [58].…”

“…In the current study, a group of 15 test genes implicated in LC-MS/MS analyses of surface sub-proteomes of L. monocytogenes as suspect adhesins were derived from (two each) strongly- and weakly-adherent phenotypic groups of L. monocytogenes ; the strains used in the current study were from the same L. monocytogenes food isolates (i.e., CW35 and 99-38) used in a prior LC-MS/MS study [24]. Using a fluorescent microplate adherence assay [18] (Table 1) eight previously characterized strains of L. monocytogenes were confirmed as belonging to two distinct adherence groups of L. monocytogenes (Figure 1 and Figure 2).…”

“…Insights on attachment mechansims may provide for more effective sanitation of food processing facilities. In this study, mRNA levels of gene transcription were evaluated for 15 genes encoding cell surface proteins identified previously as potentially involved with attachment to abiotic surfaces [24]. These results are compared to those of Chen et al [25] who evaluated two genes, inl A and inl B to establish positive correlations between gene expression and attachment strength of two adherent phenotypes of L. monocytogenes .…”

“…These results are compared to those of Chen et al [25] who evaluated two genes, inl A and inl B to establish positive correlations between gene expression and attachment strength of two adherent phenotypes of L. monocytogenes . Gene targets were determined based on multiple LC-MS/MS comparative analyses of surface sub-proteome extracts of adherence variants of L. monocytogenes (CW35/weak vs. 99-38/strong) [24]. A group of 15 genes, including a 16S rRNA reference gene [26], inl A [25], and 14 other target genes suggested in LC-MS/MS data were utilized for this purpose [24].…”

“…Gene targets were determined based on multiple LC-MS/MS comparative analyses of surface sub-proteome extracts of adherence variants of L. monocytogenes (CW35/weak vs. 99-38/strong) [24]. A group of 15 genes, including a 16S rRNA reference gene [26], inl A [25], and 14 other target genes suggested in LC-MS/MS data were utilized for this purpose [24]. …”