Abstract
AggLb is the largest (318.6 kDa) aggregation-promoting protein of Lactobacillus paracasei subsp. paracasei BGNJ1-64 responsible for forming large cell aggregates, which causes auto-aggregation, collagen binding and pathogen exclusion in vitro. It contains an N-terminus leader peptide, followed by six successive collagen binding domains, 20 successive repeats (CnaB-like domains) and an LPXTG sorting signal at the C-terminus for cell wall anchoring. Experimental information about the roles of the domains of AggLb is currently unknown. To define the domain that confers cell aggregation and the key domains for interactions of specific affinity between AggLb and components of the extracellular matrix, we constructed a series of variants of the aggLb gene and expressed them in Lactococcus lactis subsp. lactis BGKP1-20 using a lactococcal promoter. All of the variants contained a leader peptide, an inter collagen binding-CnaB domain region (used to raise an anti-AggLb antibody), an anchor domain and a different number of collagen binding and CnaB-like domains. The role of the collagen binding repeats of the N-terminus in auto-aggregation and binding to collagen and fibronectin was confirmed. Deletion of the collagen binding repeats II, III, and IV resulted in a loss of the strong auto-aggregation, collagen and fibronectin binding abilities whereas the biofilm formation capability was increased. The strong auto-aggregation, collagen and fibronectin binding abilities of AggLb were negatively correlated to biofilm formation.
Highlights
Lactobacillus strains could exhibit probiotic characteristics, which confer a variety of beneficial health effects on the host and they have a number of features that make it suitable for dairy applications (Salminen et al, 1998; Lebeer et al, 2008; Sisto and Lavermicocca, 2012; Giraffa, 2014)
The constructs (Figure 1; Table 1) were transformed into Lc. lactis subsp. lactis BGKP1-20 and expression was analyzed by Dot blot (Figure 2) using an anti-AggLb antibody raised against the transitional region covering the last part of the first region and the beginning of the second subclone of AggLb because this part is present in all of the constructs
Paracasei BGNJ1-641 and Lc. lactis subsp. lactis BGKP1-20) and between strains carrying the first part of the aggLb gene and those variants that had only two or fewer collagen binding domains; these results indicate a role of the collagen binding domains in the interaction with collagen and fibronectin, but the last 18 CnaB-like domains are not indispensable (Figures 4 and 5)
Summary
Lactobacillus strains could exhibit probiotic characteristics, which confer a variety of beneficial health effects on the host and they have a number of features that make it suitable for dairy applications (Salminen et al, 1998; Lebeer et al, 2008; Sisto and Lavermicocca, 2012; Giraffa, 2014). It was found that adhesion of lactobacilli to components of the extracellular matrix (ECM) such as mucin, fibronectin, collagen, laminin, or fibrinogen may have a direct impact on their probiotic function, e.g., in preventing the adhesion to and the colonization of damaged intestinal tissue sites by invading pathogens (Lorca et al, 2002). The ability of pathogenic bacteria to adhere to distinct components of the ECM, such as collagen and fibronectin, is enabled or facilitated by the expression of ECM-binding proteins, termed adhesins. Binding of GAS to epithelial cells involves an interaction between M protein and fibronectin (Oehmcke et al, 2010). In addition some biofilm factors related to aggregation ability, for example, Bap protein of S. aureus facilitates the persistence in the mammary gland by enhancing adhesion to epithelial cells and prevents cellular internalization through the binding to GP96 host receptor (Taglialegna et al, 2016)
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