Sortase D Forms the Covalent Bond That Links BcpB to the Tip of Bacillus cereus Pili

Self Cite

Gram-positive bacteria elaborate pili and do so without the participation of folding chaperones or disulfide bond catalysts. Sortases, enzymes that cut pilin precursors, form covalent bonds that link pilin subunits and assemble pili on the bacterial surface. We determined the x-ray structure of BcpA, the major pilin subunit of Bacillus cereus . The BcpA precursor encompasses 2 Ig folds (CNA 2 and CNA 3 ) and one jelly-roll domain (XNA) each of which synthesizes a single intramolecular amide bond. A fourth amide bond, derived from the Ig fold of CNA 1 , is formed only after pilin subunits have been incorporated into pili. We report that the domains of pilin precursors have evolved to synthesize a discrete sequence of intramolecular amide bonds, thereby conferring structural stability and protease resistance to pili.

Section: Intramolecular Amide Bonds Stabilize Pili On the Surface Of mentioning

confidence: 99%

Intramolecular amide bonds stabilize pili on the surface of bacilli

Budzik¹,

Poor

Faull

et al. 2009

Self Cite

“…Finally, the entire assembly is covalently attached to the cell wall peptidoglycan by a housekeeping sortase (9,11). Presumably, the tip pilin SpaC is linked to the SpaA shaft via the same reaction as recently identified for the tip pilin BcpB of Bacillus cereus (12). It remains unclear how the minor pilin SpaB is incorporated into the pilus structure, although recent evidence indicates that SpaB forms the basal subunit for tethering the pilus to the cell wall and that it, too, is attached to the polymer via a specific Lys residue (9).…”

mentioning

confidence: 99%

The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds

Kang

Paterson

Gaspar

et al. 2009

104

166

Cell-surface pili are important virulence factors that enable bacterial pathogens to adhere to specific host tissues and modulate host immune response. Relatively little is known about the structure of Gram-positive bacterial pili, which are built by the sortase-catalyzed covalent crosslinking of individual pilin proteins. Here we report the 1.6-Å resolution crystal structure of the shaft pilin component SpaA from Corynebacterium diphtheriae , revealing both common and unique features. The SpaA pilin comprises 3 tandem Ig-like domains, with characteristic folds related to those typically found in non-pilus adhesins. Whereas both the middle and the C-terminal domains contain an intramolecular Lys–Asn isopeptide bond, previously detected in the shaft pilins of Streptococcus pyogenes and Bacillus cereus , the middle Ig-like domain also harbors a calcium ion, and the C-terminal domain contains a disulfide bond. By mass spectrometry, we show that the SpaA monomers are cross-linked in the assembled pili by a Lys–Thr isopeptide bond, as predicted by previous genetic studies. Together, our results reveal that despite profound dissimilarities in primary sequences, the shaft pilins of Gram-positive pathogens have strikingly similar tertiary structures, suggesting a modular backbone construction, including stabilizing intermolecular and intramolecular isopeptide bonds.

“…These Gram-positive pili have a narrow diameter (1-10 nm) but can project outwardly from the cell surface with lengths of 1 m or more. Unlike the Gram-negative pili, each Gram-positive pilus is an assembly of multiple pilin subunits coupled to each other covalently by the transpeptidase activity of the pilin-specific sortase (13)(14)(15). Recent studies have established that the resulting isopeptide bonds are formed between the threonine of an LPXTG-like motif and the lysine of an YPKN pilin motif in the different pilin subunits (15,16).…”

mentioning

confidence: 99%

“…Recent studies have established that the resulting isopeptide bonds are formed between the threonine of an LPXTG-like motif and the lysine of an YPKN pilin motif in the different pilin subunits (15,16). Another membrane-bound transpeptidase, the housekeeping sortase, also recognizing the LPXTG-like motif on target pilins is responsible for attaching the base of the elongated pilus covalently to the peptidoglycan in the cell wall (13)(14)(15). Typically, the pilus is a heterotrimer composed of a major pilin forming the pilus shaft, a minor pilin decorating the pilus backbone, and another minor pilin with adhesive properties often situated at the pilus tip (13,14).…”

mentioning

confidence: 99%

“…Unlike the Gram-negative pili, each Gram-positive pilus is an assembly of multiple pilin subunits coupled to each other covalently by the transpeptidase activity of the pilin-specific sortase (13)(14)(15). Recent studies have established that the resulting isopeptide bonds are formed between the threonine of an LPXTG-like motif and the lysine of an YPKN pilin motif in the different pilin subunits (15,16). Another membrane-bound transpeptidase, the housekeeping sortase, also recognizing the LPXTG-like motif on target pilins is responsible for attaching the base of the elongated pilus covalently to the peptidoglycan in the cell wall (13)(14)(15).…”

mentioning

confidence: 99%

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Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein

Kankainen

Paulín

Tynkkynen

et al. 2009

649

901

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.genome ͉ probiotics ͉ adhesion ͉ pilus ͉ lactic acid bacteria