Human Gut-Commensalic Lactobacillus ruminis ATCC 25644 Displays Sortase-Assembled Surface Piliation: Phenotypic Characterization of Its Fimbrial Operon through In Silico Predictive Analysis and Recombinant Expression in Lactococcus lactis

Abstract

Sortase-dependent surface pili (or fimbriae) in Gram-positive bacteria are well documented as a key virulence factor for certain harmful opportunistic pathogens. However, it is only recently known that these multi-subunit protein appendages are also belonging to the “friendly” commensals and now, with this new perspective, they have come to be categorized as a niche-adaptation factor as well. In this regard, it was shown earlier that sortase-assembled piliation is a native fixture of two human intestinal commensalics (i.e., Lactobacillus rhamnosus and Bifidobacterium bifidum), and correspondingly where the pili involved have a significant role in cellular adhesion and immunomodulation processes. We now reveal that intestinal indigenous (or autochthonous) Lactobacillus ruminis is another surface-piliated commensal lactobacillar species. Heeding to in silico expectations, the predicted loci for the LrpCBA-called pili are organized tandemly in the L. ruminis genome as a canonical fimbrial operon, which then encodes for three pilin-proteins and a single C-type sortase enzyme. Through electron microscopic means, we showed that these pilus formations are a surface assemblage of tip, basal, and backbone pilin subunits (respectively named LrpC, LrpB, and LrpA) in L. ruminis, and also when expressed recombinantly in Lactococcus lactis. As well, by using the recombinant-piliated lactococci, we could define certain ecologically relevant phenotypic traits, such as the ability to adhere to extracellular matrix proteins and gut epithelial cells, but also to effectuate an induced dampening on Toll-like receptor 2 signaling and interleukin-8 responsiveness in immune-related cells. Within the context of the intestinal microcosm, by wielding such niche-advantageous cell-surface properties the LrpCBA pilus would undoubtedly have a requisite functional role in the colonization dynamics of L. ruminis indigeneity. Our study provides only the second description of a native-piliated Lactobacillus species, but at the same time also involves the structural and functional characterization of a third type of lactobacillar pilus.

Pili in Probiotic Bacteria

“…Since then, it has been identified in several species and strains of probiotic and other commensal bacteria by genomic analysis and shown to be essential for their adherence and colonization in GIT. Their presence was further confirmed by imaging analysis in the L. rhamnosus GG [29,31], genus of Bifidobacterium [32,33], Lacococcus lactis IL1403 and TIL448 [34,35], and recently in Lactobacillus ruminis ATCC 25644 [36]. Hence, the view of surface piliation has now been expanded to include its role also as a niche-adaptation factor.…”

A Comparative Characterization of Different Host-sourced Lactobacillus ruminis Strains and Their Adhesive, Inhibitory, and Immunomodulating Functions

“…Rather instead, L. ruminis is more likely to inhabit the epithelial cell zone of the gut, as it has the capacity to bind ECM proteins, primarily by the adhesiveness of its surface piliation, lrpCBA - encoded pili in case of strain ATCC 25644 (Yu X. et al, 2015), and we presume that the binding behavior of the other three L. ruminis strains is also pilus-dependent. The lrpCBA pilus operon has been verified by PCR in ATCC 27780 and ATCC 27781 and, based on genomic data, at least one of the genes encodes a pilin protein with a predicted collagen-binding domain (our unpublished data).…”

SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis

“…Whilst not a universal cell-surface attribute of all Grampositive bacteria, the long and limb-like sortase-dependent pilus can nonetheless be found in a wide variety of different genera and species, including both pathogens (Khare & Narayana, 2017;Pansegrau & Bagnoli, 2017;Telford et al, 2006;Ton-That & Schneewind, 2004) and intestinal commensals (Kankainen et al, 2009;Lebeer et al, 2009;Turroni et al, 2013;Yu et al, 2015). This non-flagellar but adhesive appendage allows bacterial cells to outcompete non-piliated forms for host-environment receptor sites, and thereby acts as an effective colonization factor for virulence activity or ecological niche adaptation (Danne & Dramsi, 2012;Krishnan et al, 2016;.…”