Abstract
Eleven Lactobacillus strains with strong aggregation abilities were selected from a laboratory collection. In two of the strains, genes associated with aggregation capability were plasmid located and found to strongly correlate with collagen binding. The gene encoding the auto-aggregation-promoting protein (AggLb) of Lactobacillus paracasei subsp. paracasei BGNJ1-64 was cloned using a novel, wide-range-host shuttle cloning vector, pAZILSJ. The clone pALb35, containing a 11377-bp DNA fragment, was selected from the SacI plasmid library for its ability to provide carriers with the aggregation phenotype. The complete fragment was sequenced and four potential ORFs were detected, including the aggLb gene and three surrounding transposase genes. AggLb is the largest known cell-surface protein in lactobacilli, consisting of 2998 aa (318,611 Da). AggLb belongs to the collagen-binding superfamily and its C-terminal region contains 20 successive repeats that are identical even at the nucleotide level. Deletion of aggLb causes a loss of the capacity to form cell aggregates, whereas overexpression increases cellular aggregation, hydrophobicity and collagen-binding potential. PCR screening performed with three sets of primers based on the aggLb gene of BGNJ1-64 enabled detection of the same type of aggLb gene in five of eleven selected aggregation-positive Lactobacillus strains. Heterologous expression of aggLb confirmed the crucial role of the AggLb protein in cell aggregation and specific collagen binding, indicating that AggLb has a useful probiotic function in effective colonization of host tissue and prevention of pathogen colonization.
Highlights
Due to their long history of safe use in food fermentation and preservation, lactobacilli currently carry the ‘Qualified Presumption of Safety’ (QPS) status [1]
Because exhibition of the auto-aggregation phenotype should be related to the surface proteins, the susceptibility of the aggregation determinants to protease inactivation was tested by incubation of bacterial cells with proteinase K
The main goal of this study was to investigate possible role of Lactobacillus aggregation factors in determination of features favored in probiotics, such as collagen binding and protection from adhesion of pathogenic bacteria
Summary
Due to their long history of safe use in food fermentation and preservation, lactobacilli currently carry the ‘Qualified Presumption of Safety’ (QPS) status [1]. Probiotic microorganisms express cell-surface adhesins that mediate microbial adhesion to the extracellular matrix (ECM) components of host tissue such as mucin, fibronectin, collagen, laminin or fibrinogen [5]. Various human pathogenic bacteria exhibit specific adhesiveness to collagenous proteins [8, 9]. This interaction is critical in early-phase infection, and is strongly related to the virulence of the pathogen. Through the action of cell-surface adhesins, pathogens successfully interact with proteins of the ECM, preserving peristalsis and enabling colonization of the tissue and infection [9]. An example is the collagen-binding protein that enables Staphylococcus aureus cells to adhere to cartilage in vitro [10] and which has been described as a major virulence factor
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