Phenotypical Analysis of the Lactobacillus rhamnosus GG Fimbrial spaFED Operon: Surface Expression and Functional Characterization of Recombinant SpaFED Pili in Lactococcus lactis

Abstract

A noticeable genomic feature of many piliated Gram-positive bacterial species is the presence of more than one pilus-encoding operon. Paradigmatically, the gut-adapted Lactobacillus rhamnosus GG strain contains two different fimbrial operons in its genome. However, whereas one of these operons (called spaCBA) is encoding for the functionally mucus-/collagen-binding SpaCBA pilus, for the other operon (called spaFED) any native expression of the SpaFED-called pili is still the subject of some uncertainty. Irrespective of such considerations, we decided it would be of relevance or interest to decipher the gross structure of this pilus type, and as well assess its functional capabilities for cellular adhesion and immunostimulation. For this, and by following the approach we had used previously to explicate the immuno-properties of SpaCBA pili, we constructed nisin-inducible expression clones producing either wild-type or SpaF pilin-deleted surface-assembled L. rhamnosus GG SpaFED pili on Lactococcus lactis cells. Using these piliated lactococcal constructs, we found that the pilin-polymerized architecture of a recombinant-produced SpaFED pilus coincides with sequence-based functional predictions of the related pilins, and in fact is prototypical of those other sortase-dependent pilus-like structures thus far characterized for piliated Gram-positive bacteria. Moreover, we confirmed that among the different pilin subunits encompassing spaFED operon-encoded pili, the SpaF pilin is a main adhesion determinant, and when present in the assembled structure can mediate pilus binding to mucus, certain extracellular matrix proteins, and different gut epithelial cell lines. However, somewhat unexpectedly, when recombinant SpaFED pili are surface-attached, we found that they could not potentiate the existing lactococcal cell-induced immune responses so elicited from intestinal- and immune-related cells, but rather instead, they could dampen them. Accordingly, we have now provided the first phenotypical description of a spaFED pilus operon, and with that furthered the functional understanding of surface piliation for a particular gut-commensalic genre of piliated Gram-positive bacteria.

Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster

“…rhamnosus GG also carries a pilus gene cluster, spaFED , on its chromosome, where SpaD is the pilus backbone protein, SpaE is at the base of the pilus structure, and SpaF is the minor pilin subunit that locates to the tip of the pilus. SpaF was also shown to be responsible for adhesion of pili-carrying cells to the intestinal mucus [42,88]. Furthermore, cloning of spaC GG , spaB GG , spaD GG , spaE GG , spaF GG in Escherichia coli and assessment of the adherence of these proteins to human intestinal mucus reveled that also the SpaB pilin subunit plays a role in binding to intestinal mucus, through electrostatic contacts [42].…”

Tackling probiotic and gut microbiota functionality through proteomics

“…acidophilus [143] or fimbriae in Lb. rhamnosus [144] are remarkable and have been identified using proteomic approaches. In this sense, proteomics has been relevant in the identification a high number of moonlighting proteins, proteins that are secreted without harbouring any signal peptide.…”

Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adaptedLactobacillus rhamnosusGG

“…Among the latter is Lactobacillus rhamnosus GG, a gut-adapted strain that has been advocated to have various health benefits (Gorbach, 2000) and the genome of which contains operons (spaCBA and spaFED) that encode two different types of sortase-dependent pili (Kankainen et al, 2009). One of these pilus types is called SpaCBA and is made up of the tip SpaC, basal SpaB and backbone SpaA pilins (Reunanen et al, 2012), whereas the other is called SpaFED and is comprised of the tip SpaF, basal SpaE and backbone SpaD pilins (Rintahaka et al, 2014). Owing to the constitutive expression of the fimbrial spaCBA operon in L. rhamnosus GG, the SpaCBA pilus is generally better studied than the SpaFED pilus, the operon for which shows no native expression under laboratory conditions (Reunanen et al, 2012).…”

“…Nonetheless, the spaFED genes were recently cloned in Lactococcus lactis to produce a recombinant form of the SpaFED pilus ( Figs. 1a and 1b), which underwent functional and structural characterization (Rintahaka et al, 2014). In terms of function, both pilus types can bind to intestinal mucus, an ability attributed to the SpaC, SpaB and SpaF ancillary pilins (Kankainen et al, 2009;von Ossowski et al, 2010).…”