An extracellular Serine/Threonine-rich protein from Lactobacillus plantarum NCIMB 8826 is a novel aggregation-promoting factor with affinity to mucin

Abstract

Autoaggregation in lactic acid bacteria is directly related to the production of certain extracellular proteins, notably, aggregation-promoting factors (APFs). Production of aggregation-promoting factors confers beneficial traits to probiotic-producing strains, contributing to their fitness for the intestinal environment. Furthermore, coaggregation with pathogens has been proposed to be a beneficial mechanism in probiotic lactic acid bacteria. This mechanism would limit attachment of the pathogen to the gut mucosa, favoring its removal by the human immune system. In the present paper, we have characterized a novel aggregation-promoting factor in Lactobacillus plantarum. A mutant with a knockout of the D1 gene showed loss of its autoaggregative phenotype and a decreased ability to bind to mucin, indicating an adhesion role of this protein. In addition, heterologous production of the D1 protein or an internal fragment of the protein, characterized by its abundance in serine/threonine, strongly induced autoaggregation in Lactococcus lactis. This result strongly suggested that this internal fragment is responsible for the bioactivity of D1 as an APF. To our knowledge, this is the first report on a gene coding for an aggregation-promoting factor in Lb. plantarum.

Fig 1

Schematic representation of D1 gene deletion by homologous recombination using plasmid pMN18. E, erythromycin; Cm, Ery, and Amp, chloramphenicol, erythromycin, and ampicillin resistance cassettes, respectively.

Fig 2

(A) SDS-PAGE analysis of extracellular proteins from wild-type strain Lb. plantarum NCIMB 8826 and the mutant strain Lb. plantarum NCIMB 8826ΔD1, showing the absence of protein D1 in the latter. Lane MM, molecular marker; lane wt, wild type. (B) Rates of Lb. plantarum NCIMB 8826 (black boxes) and Lb. plantarum NCIMB 8826ΔD1 (white boxes) adhesion to mucin type III, mucin type II, and fibronectin. ***, P < 0.001. (C) Loss of the autoaggregative phenotype in the NCIMB 8826ΔD1 strain after 24 h in PBS buffer. White arrow, unaggregated bacteria that remained in suspension.

Fig 3

(A) Photographs of culture turbidity of induced (with nisin [Nis]) or uninduced (−) 24-h cultures of Lc. lactis strains D1 and ST, showing the strong aggregative phenotype of the latter when the peptide ST is produced. (B) Growth curves of strain NZ9000 harboring the empty plasmid pNZ8110, strain D1, and strain ST induced or not with nisin (the induction point is marked with an arrow). The A₆₀₀ of the cultures was measured over time without agitation and was used as a measure of the rate of aggregation of the bacteria. (C) Photographs of culture turbidity of induced (with nisin [Nis]) and uninduced (−) 24-h cultures of strain ST showing the clumps that originated after bacterial aggregation.

Fig 4

Adhesion of recombinant Lc. lactis strains to mucin type III. White bars, adhesion in nisin (Nis)-induced cultures; black bars, adhesion in uninduced cultures. D1 and ST refer to the corresponding Lactococcus strains expressing the D1 gene or the ST-rich fragment, as indicated in Table 1. Lc. lactis harboring the empty plasmid pNZ8110 was used as a negative control. ***, P < 0.001.