Crystal structure of lactobacillar SpaC reveals an atypical five-domain pilus tip adhesin: Exposing its substrate-binding and assembly in SpaCBA pili

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Highlights

  • Structural insights about the adhesive SpaC tip pilin subunit of the L. rhamnosus GG sortase-dependent SpaCBA pilus are revealed.

  • Crystal structure of full-length SpaC was determined at 1.9 Å resolution.

  • SpaC structure displays an atypical five-domain arrangement.

  • Structural basis for the multi-substrate binding nature of SpaC is provided.

  • Structural model for the localized assembly of SpaC at the pilus tip is proposed.

Abstract

Adhesion to cell surfaces is an essential and early prerequisite for successful host colonization by bacteria, and in most instances involves the specificities of various adhesins. Among bacterial Gram-positives, some genera and species mediate attachment to host cells by using long non-flagellar appendages called sortase-dependent pili. A case in point is the beneficial Lactobacillus rhamnosus GG gut-adapted strain that produces the so-called SpaCBA pilus, a structure noted for its promiscuous binding to intestinal mucus and collagen. Structurally, SpaCBA pili are heteropolymers of three different pilin-protein subunits, each with its own location and function in the pilus: backbone SpaA for length, basal SpaB for anchoring, and tip SpaC for adhesion. Previously, we solved the SpaA tertiary structure by X-ray crystallography and also reported on the crystallization of SpaB and SpaC. Here, we reveal the full-length high-resolution (1.9 Å) crystal structure of SpaC, a first for a sortase-dependent pilus-bearing commensal. The SpaC structure, unlike the representative four-domain architecture of other Gram-positive tip pilins, espouses an atypically longer five-domain arrangement that includes N-terminal ‘binding’ and C-terminal ‘stalk’ regions of two and three domains, respectively. With the prospect of establishing new mechanistic insights, we provide a structural basis for the multi-substrate binding nature of SpaC, as well as a structural model that reconciles its exclusive localization at the SpaCBA pilus tip.

Introduction

Sortase-dependent pili are non-flagellar proteinaceous structures found exclusively on the outer surface of certain genera and species of Gram-positive bacteria (Khare and Narayana, 2017, Pansegrau and Bagnoli, 2017, Telford et al., 2006, Ton-That and Schneewind, 2004, von Ossowski, 2017). By having a long and limb-like morphology, as well as a strongly adhesive character, this type of pilus formation helps establish the very earliest bacterial contact and adhesion to host cells and tissues, which in effect confers a clear survival benefit to so-piliated microbes. Consequently, the Gram-positive sortase-dependent pilus is well recognized as a key competitive colonization advantage for promoting virulence activity (pathogens) and niche adaptation (non-pathogens) (Danne and Dramsi, 2012, Krishnan et al., 2016, von Ossowski, 2017). From an architectural standpoint, the sortase-dependent pilus is simply comprised of a head-to-tail linear assembly of tip, backbone, and basal protein subunits (pilins) (Hilleringmann et al., 2009). For such a pilus arrangement, each pilin has a specific structural location and function, with the backbone pilin providing polymeric length, and the ancillary tip and basal pilins facilitating adhesion and anchoring, respectively. The polymerization of backbone pilins into an elongated pilus structure is mediated through the transpeptidase activity of a pilin-specific C-type sortase (Hendrickx et al., 2011, Mandlik et al., 2008b, Siegel et al., 2016). This enzyme catalyzes the formation of a covalent isopeptide bond between two conserved residues within different peptide regions in each adjacent pilin subunit. Here, this involves the so-called ‘linking’ lysine (K) in the YPKN pilin motif at the N-terminus of one pilin (head) and a threonine (T) in the C-terminal sorting signal LPXTG motif of another pilin (tail). Interestingly, as these two peptide motifs co-exist in the basal pilins, this type of subunit is occasionally also incorporated within the polymeric pilus backbone (Mandlik et al., 2008b). However, regarding the tip pilin subunit, it just possesses the LPXTG pentapeptide and, due to this, is limited to only the outer peripheral end of the pilus structure (Hilleringmann et al., 2009). Since the genes for the tip, backbone, and basal pilins and the C-type sortase are always found clustered together as an operon in the genome, this ensures synchronized protein production and availability during the pilus polymerization process (Mandlik et al., 2008b). At some point when further assembly of the pilin subunits no longer continues, the catalytic activity from a different sortase (housekeeping A-type) fastens the lengthy pilus to the cell wall through covalent bonding with the LPXTG-threonine of the lastly incorporated basal pilin (Chang et al., 2019, Mandlik et al., 2008a).

Lactobacillus rhamnosus GG, a human gut-adapted and health-benefiting probiotic strain (Pace et al., 2015), is the first and best studied representative of Gram-positive commensals that constitutively produce sortase-dependent pili (Kankainen et al., 2009, von Ossowski, 2017). So-called SpaCBA pili are encoded by the spaCBA operon (spaC-spaB-spaA-srtC1) and built up from the tip SpaC, basal SpaB, and backbone SpaA pilin subunits (Kankainen et al., 2009, Kant et al., 2014). Incidentally, L. rhamnosus GG also encodes another pilus operon called spaFED (spaF-spaE-spaD-srtC2) (Kankainen et al., 2009, Kant et al., 2014), but while its native expression is yet to be established under testing conditions (Reunanen et al., 2012), a fully assembled recombinant form of the SpaFED pilus has been produced in Lactococcus lactis (Rintahaka et al., 2014). In the quest to reveal the molecular aspects governing gut adaptation and probiosis in L. rhamnosus GG, it was uncovered early on that the SpaCBA pilus can bind to human-sourced intestinal mucus via the SpaC tip subunit (Kankainen et al., 2009, von Ossowski et al., 2010, von Ossowski et al., 2013). Accordingly, the SpaC pilus protein came to be regarded as one of the main adhesion determinants in L. rhamnosus GG, insofar as its mucoadhesiveness presumably contributes to the prolonged transient gut persistence of this allochthonous strain (Kankainen et al., 2009). Functionally, as the predicted primary structure of SpaC contains the adhesive von Willebrand Factor A (vWFA) domain, which itself also includes a small segment with partial homology to a fucose-binding lectin domain (Kankainen et al., 2009), this tip pilin might use a lectin-type recognition mechanism for adhering to, e.g., glycosylated mucin protein, the major component of colonic mucus. Further support for such SpaC-glycan contact comes from a subsequent study that reported binding between the SpaC subunit and mucin-derived oligosaccharide chains, or more specifically via the non-reducing end β-galactoside moiety (Nishiyama et al., 2016). Interestingly, another study revealed that the SpaC pilin has a promiscuous binding nature and thus, despite the absence of any dedicated collagen-binding domains, can also adhere to ECM collagen (Tripathi et al., 2013). Because the interaction of SpaC with collagen seems to dissociate rapidly, there is some speculation suggesting that this would permit the SpaCBA pilus to quickly detach/attach from host cells in the gut epithelium, particularly in damaged areas where ECM proteins become accessible to bacteria (Tripathi et al., 2013). Related to this, additional studies have indicated that the SpaCBA pilus also utilizes its SpaC subunit to promote adherence between L. rhamnosus GG and intestinal epithelial cells (Ardita et al., 2014, Lebeer et al., 2012). Still further, other studies have associated the adhesiveness of SpaC with the tendency of SpaCBA pili to self-adhere and bundle together as a larger macromolecular unit (Tripathi et al., 2012, Tripathi et al., 2013). Purportedly, this might contribute to the reported SpaC-dependent cell-aggregation and biofilm growth of piliated L. rhamnosus GG (Lebeer et al., 2012). What is quite surprising, however, is that the findings from immuno-electron microscopy (EM) studies give the impression that the SpaC tip pilin is also impregnated throughout the polymerized backbone of SpaA pilins (Kankainen et al., 2009, Reunanen et al., 2012, von Ossowski et al., 2013). Presumably, this is further reflected in the role of SpaCBA pili in L. rhamnosus GG as an indispensable host-colonization adhesin.

To elucidate the structural basis for the unique and versatile functional properties of the SpaC tip pilin, we undertook to produce protein crystals (Kant et al., 2016) and solve its tertiary structure by X-ray crystallography. As part of our earlier work, we had already begun crystallographic studies of the pilins (backbone and basal) and sortases (Chaurasia et al., 2015, Kumar Megta et al., 2019, Mishra et al., 2017, Pratap et al., 2019, Singh et al., 2013) that encompass the L. rhamnosus GG SpaCBA and SpaFED pili. Based on the solved crystal structures of SpaA, SpaD, and SpaE (Chaurasia et al., 2016, Chaurasia et al., 2018, Megta et al., 2019), each shares a close structural and modular resemblance with the typical Gram-positive pilin and is comprised of the CnaA and CnaB domains (Kang and Baker, 2012, Krishnan, 2015), i.e., modified immunoglobulin (Ig)-like folds of the staphylococcal collagen adhesin (Cna) (Deivanayagam et al., 2000, Symersky et al., 1997). Here, the backbone SpaA (Chaurasia et al., 2016) and basal SpaE (Megta et al., 2019) pilins are both two-domain proteins (CnaB-CnaB), whereas the larger backbone SpaD pilin consists of three domains (CnaB-CnaA-CnaB) (Chaurasia et al., 2018). The common topology of these CnaA and CnaB domains is a core fold of nine and seven β-strands, respectively (Vengadesan and Narayana, 2011), added to which are some extra structural topologies for each particular pilin (Chaurasia et al., 2016, Chaurasia et al., 2018, Megta et al., 2019). Consistent with other Gram-positive pilus proteins (Kang and Baker, 2009, Kang et al., 2007), as both domain folds can be considered structurally reinforced and rigid due to the autocatalytic formation of internal isopeptide bonds between lysine and asparagine/aspartate (K–N/D) residues, this phenomenon, though with some unique characteristics, is also found in SpaA, SpaD, and SpaE (Chaurasia et al., 2016, Chaurasia et al., 2018, Megta et al., 2019).

Success in obtaining X-ray structures of Gram-positive pilus adhesins often remains a problematic task, mainly owing to their large size, flexible nature, multi-domain arrangement, and complicated folding pattern. Hence, there are only a limited number of solved structures for the tip pilins, either as full-length protein (RrgA from Streptococcus pneumoniae (Izore et al., 2010)) or as domain fragments (GBS104 from Streptococcus agalactiae (Krishnan et al., 2013) as well as Cpa and Spy0125 from Streptococcus pyogenes (Linke-Winnebeck et al., 2014, Pointon et al., 2010)). Nonetheless, a representative structure of this pilus subunit has emerged and consists of four domains, i.e., a globular substrate-binding domain attached onto a stalk-like base of three CnaA/CnaB domains. As for the L. rhamnosus GG SpaC tip pilin, we now report on its crystal structure at a 1.9 Å resolution. Remarkably, SpaC possesses a longer five-domain arrangement, which is further organized into so-called ‘binding’ and ‘stalk’ regions. Herein, we also provide some mechanistic insights into the binding of host surfaces by the adhesive SpaC subunit as well as describe a structural model for incorporating SpaC onto the tip of the SpaCBA pilus.

Section snippets

Protein expression and purification

Full-length (residues 36–856) recombinant GG-SpaC (GG-SpaCFL) protein derived from the Lactobacillus rhamnosus GG (ATCC 53103) spaC gene was cloned and produced in Escherichia coli BL21 (DE3) pLysS as described previously (Kant et al., 2016). GG-SpaCFL protein also includes seven cloning vector residues (MGRDPNS) in place of its N-terminal signal peptide, along with a hexahistidine (LEHHHHHH) tag and part of the LPXTG motif sorting signal at the C-terminus. Selenomethionine substituted

GG-SpaC adopts an atypical five-domain structure

Full-length recombinant SpaC from L. rhamnosus GG (GG-SpaCFL) was produced in E. coli (Kant et al., 2016) and subsequently crystallized (Kant et al., 2016) (see further in Material and methods). The crystal structure of GG-SpaCFL revealed an overall architecture of five domains (ordered top-to-bottom as D2-D1-D3-D4-D5) that is grouped functionally into the N-terminal binding and C-terminal stalk regions (Fig. 1A–F and Table 1). Outwardly projecting from the top end of the binding region is the

Conclusions

The view that gut-friendly and advocated probiotics like L. rhamnosus GG help promote digestive well-being is amply documented by a multitude of clinical and laboratory studies. Even so, there remains scant structural information on the molecular mechanisms that underlie these beneficial effects. As persistence and survival in the intestinal tract are often linked to how long one can expect these benefits to last, the adhesion capacity of probiotic strains is an obvious criterion for their

Accession code

Atomic coordinates for the crystal structures of GG-SpaC were deposited in the PDB under the following accession numbers: 6M3Y (GG-SpaCFL), 6M48 (GG-SpaCFL-A&B), 6M7C (native GG-SpaCD4&D5), and 7BVX (iodide-derivatized GG-SpaCD4&D5).

Contributions

A.K. A.P., I.v.O., and V.K. designed the study; A.K., I.v.O., and V.K. performed experiments and analyzed data; A.K. and V.K. wrote initial draft; V.K. and I.v.O. revised the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This project was supported by the Regional Centre for Biotechnology (RCB) and the Department of Biotechnology (DBT) (Grant No. BT/PR5891/BRB/10/1098/2012), India. We thank Drs. Hassan Belrhali and Babu Manjashetty (BM14 beamline, ESRF), Dr. Nicolas Foos (ID23 Beamline, ESRF), and Drs. Babu Manjashetty and Annie Heroux (XRD2 beamline, Elettra) for their help during data collection. Access to synchrotron facilities was supported by DBT-ESRF and DST-Elettra collobrative projects. We also

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