Characterization of the SpaCBA Pilus Fibers in the Probiotic Lactobacillus rhamnosus GG

Abstract

cLactobacillus rhamnosus GG is a human intestinal isolate that has been studied intensively because of its probiotic properties. We have previously shown that L. rhamnosus GG produces proteinaceous pili that earlier had been observed only in Gram-positive pathogens (M. Kankainen et al., Proc. Natl. Acad. Sci. U. S. A. 106:17193-17198, 2009). These pili were found to be encoded by the spaCBA gene cluster, and the pilus-associated SpaC pilin was shown to confer on the cells a mucus-binding ability. In addition to the spaCBA cluster, another putative pilus cluster, spaFED, was predicted from the L. rhamnosus GG genome sequence. Herein, we show that only SpaCBA pili are produced by L. rhamnosus, and we describe a detailed analysis of cell wall-associated and affinity-purified SpaCBA pili by Western blotting and immunogold electron microscopy. Our results indicate that SpaCBA pili are heterotrimeric protrusions with a SpaA subunit as the shaft-forming major pilin. Only a few SpaB subunits could be observed in pilus fibers. Instead, SpaB pilins were found at pilus bases, as assessed by immunogold double labeling of thin sections of cells, suggesting that SpaB is involved in the termination of pilus assembly. The SpaC adhesin was present along the whole pilus length at numbers nearly equaling those of SpaA. The relative amount and uniform distribution of SpaC within pili not only makes it possible to exert both long-distance and intimate contact with host tissue but also provides mucus-binding strength, which explains the prolonged intestinal residency times observed for L. rhamnosus GG compared to that of nonpiliated lactobacilli.

Lactobacillus spp. belonging to the Casei group display a variety of adhesins

“…Isolates in which the expression of the SpaCBA complex was detected should exhibit the ability to adhere to the intestinal mucus due to the presence of the mucus-binding domain on the SpaC subunit, and to Caco-2 cells (Reunanen et al, 2012). This dependence may be observed in five out of six isolates characterized by very efficient adhesion to Caco-2 cells, mucus and HeLa cells.…”

“…This could be caused by the lack of SpaB, a protein determining the attachment of pili to the cell wall. SpaB is thought to act as a molecular switch that terminates pilus polymerization and predestines mature pili to be covalently attached to the peptidoglycan by the housekeeping sortase (Reunanen et al, 2012).…”

Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis

“…Transmission Electron Microscopy (TEM) Analysis-Stationary phase bacterial cells were immunolabeled using rabbit polyclonal anti-SpaA antibodies and fixed onto Formvar carboncoated copper grids as previously described (21). Grids were examined using JEM-1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan).…”

Lactobacillus strains belonging to Casei group display various adherence to enterocytes and mucus

“…They form a polymeric fi mbriae, which participation in the process of adhesion to the intestinal epithelium has been previously demonstrated. They also have the ability to bind to the mucus, through the presence of the subunit SpaC mucus binding domain (Kankainen et al, 2009;Reunanen et al, 2012). Another factor differentiating ability of individual strains to adhere to the intestinal epithelium are variations in the sequences encoding the fi mbrial protein structure (Toh et al, 2013) and its expression Rasinkangas et al, 2014).…”

“…Adhesion to intestinal mucus occurs with the participation of adhesins such as mucin binding proteins (Roos and Jonsson, 2002), or certain polysaccharides associated with the bacterial cell wall or secreted into the external environment (Darilmazl et al, 2011). SpaCBA protein complex forming fi mbriae on the surface of bacterial cells is responsible for the adhesion to both mucus and enterocytes (Kankainen et al, 2009;Reunanen et al, 2012). The adhesion effi ciency of different strains may show considerable differences, affecting the effi ciency of benefi cial effect of the probiotic.…”