Effects of the peptide pheromone plantaricin A and cocultivation with Lactobacillus sanfranciscensis DPPMA174 on the exoproteome and the adhesion capacity of Lactobacillus plantarum DC400

Abstract

This study aimed at investigating the extracellular and cell wall-associated proteins (exoproteome) of Lactobacillus plantarum DC400 when cultivated on modified chemically defined medium (CDM) supplemented with the chemically synthesized pheromone plantaricin A (PlnA) or cocultured with L. plantarum DPPMA20 or Lactobacillus sanfranciscensis DPPMA174. Compared to monoculture, two-dimensional gel electrophoresis (2-DE) analysis showed that the exoproteome of L. plantarum DC400 was affected by PlnA and cocultivation with strains DPPMA20 and, especially, DPPMA174. The highest similarity of the 2-DE maps was found between DC400 cells cultivated in monoculture and in coculture with strain DPPMA20. Almost all extracellular proteins (22 spots) and cell wall-associated proteins (40 spots) which showed decreased or increased levels of synthesis during growth in CDM supplemented with PlnA and/or in coculture with strain DPPMA20 or DPPMA174 were identified. On the basis of the sequences in the Kyoto Encyclopedia of Genes and Genomes database, changes to the exoproteome concerned proteins involved in quorum sensing (QS), the transport system, stress response, carbohydrate metabolism and glycolysis, oxidation/reduction processes, the proteolytic system, amino acid metabolism, cell wall and catabolic processes, and cell shape, growth, and division. Cultivation with PlnA and cocultivation with strains DPPMA20 and, especially, DPMMA174 markedly increased the capacity of L. plantarum DC400 to form biofilms, to adhere to human Caco-2 cells, and to prevent the adhesion of potential intestinal pathogens. These phenotypic traits were in part related to oversynthesized moonlighting proteins (e.g., DnaK and GroEL, pyruvate kinase, enolase, and glyceraldehyde-3-phosphate dehydrogenase) in response to QS mechanisms and interaction with L. plantarum DPPMA20 and, especially, L. sanfranciscensis DPPMA174.

Fig 1

PermutMatrixEN analysis of the amounts of proteins of Lactobacillus plantarum DC400 cells grown until the early stationary phase (16 h) was reached. Results are for monocultures of L. plantarum DC400 on modified CDM (DC400) or on CDM supplemented with 2.5 μg/ml of plantaricin A (DC400/PlnA) and cocultures of L. plantarum DC400 with L. plantarum DPPMA20 (DC400/DPPMA20) or Lactobacillus sanfranciscensis DPPMA174 (DC400/DPPMA174). Changes in protein amounts (average of three replicates) are represented colorimetrically, with red and green indicating the highest and lowest values of the standardized data, respectively, for each protein under different culture conditions. All data are shown as a percentage of dissimilarity using Euclidean distance. Protein names and spot numbers correspond to those in Tables 1 and 2. -E, extracellular proteins.

Fig 2

Schematic representation of the changes in the exoproteome (grouped on the basis of KEGG functions) of Lactobacillus plantarum DC400 cells grown until early stationary phase (16 h) was reached. Compared to monoculture, changes (increases and decreases) in the amounts of proteins (average of three replicates) are represented as follows: DC400 cells grown in CDM supplemented with 2.5 μg/ml of plantaricin A ( or ) or in coculture with L. plantarum DPPMA20 ( or ) or Lactobacillus sanfranciscensis DPPMA174 ( or ). Compared to the amounts of proteins in monoculture, the absence of changes in the amounts of proteins (average of three replicates) is represented with a minus sign. Protein names and spot numbers correspond to those in Tables 1 and 2. -E, extracellular proteins.

Fig 3

In vitro biofilm formation of Lactobacillus plantarum DC400 cultivated in monoculture on modified CDM (DC400) or in CDM supplemented with 2.5 μg/ml of plantaricin A (DC400/PlnA) or cocultured with L. plantarum DPPMA20 (DC400/DPPMA20) or Lactobacillus sanfranciscensis DPPMA174 (DC400/DPPMA174) until early stationary phase was reached (16 h at 30°C). Biofilm formation was assayed on polystyrene pegs containing ca. 200 μl of CDM alone (negative control) or CDM with DC400 cells incubated for 72 h at 30°C. The data shown are representative of at least three independent experiments. Columns with different letters indicate significant (P < 0.05) differences in biofilm formation between the different conditions assayed, according to Tukey's test.