Pilus hijacking by a bacterial coaggregation factor critical for oral biofilm development

Reardon-Robinson, Melissa E.; Wu, Chenggang; Mishra, Ashish Kumar; Chang, Chia‐Wei; Bier, Naomi; Das, Asis; Ton‐That, Hung

doi:10.1073/pnas.1321417111

Cited by 49 publications

(78 citation statements)

References 26 publications

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“…There are several limitations inherent to the mechanical study of purified pili. Pili do not exclusively contain the SpaA shaft proteins, and measurements may be confounded by incorporated adhesins and other ancillary proteins (13). Moreover, the attachment of pili to the surface and cantilever relies on nonspecific interactions, such that the vector of the force is not explicitly defined.…”

Section: Resultsmentioning

confidence: 99%

“…3E). The native FimA pili of A. oris measure 1-2 μm in length, the equivalent of ∼140-280 pilins polymerized in series, each ∼7 nm from the intermolecular cross-link to the C terminus (13). Without unfolding, such as in the CnaB Spy0128-type pilus of S. pyogenes, force extension follows a worm-like chain contour that rises unimpeded into the nanonewton range, where the lifetimes of covalent bonds are brief (5).…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, native pilus lengths vary widely. FimA pili are easily observed by EM in WT A. oris, and can exceed 2 μm in length (13). Although other Gram-positive bacteria have shorter pili, the expression of genes responsible for pilus biogenesis may be up-regulated to produce longer pili (31,32).…”

Section: Discussionmentioning

confidence: 99%

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CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

Echelman

Alegre-Cebollada

Badilla

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker intermediates that recover strength with time or ligand binding. Based on the high force extensibility, CnaA-containing pili can dissipate ∼28-fold as much energy compared with their inextensible counterparts before reaching forces sufficient to cleave covalent bonds. We propose that efficient mechanical energy dissipation is key for sustained bacterial attachment against mechanical perturbations.bacterial adhesion | mechanical stability | single-molecule force spectroscopy | Gram-positive pili | isopeptide bond B acterial infections of solid tissues begin with the attachment of bacteria to target surfaces. In many instances, bacteria adhere against forces that oppose such attachment: micturition in the genitourinary tract (1) or mucociliary flow in the respiratory tract (2), for example. In such environments, a completely immobile adherent bacterium experiences a drag force that can be approximated by Stokes law, F = 6·π·r·η·v, where r is the Stokes radius of the bacterium (∼0.5 μm), η is the viscosity of the fluid (in the respiratory mucus, 1-100 Pa·s −1 ) (3), and v is the velocity of the fluid surrounding the bacterium (Fig. 1A). Under normal mucociliary flow (1-100 μm·s −1 ) (4), forces on a single bacterium can exceed several nanonewtons. Such high forces are sufficient to cleave covalent bonds within the initial adherence structures (5), which would terminate attachment. Understanding how bacteria manage to remain attached under such strong mechanical perturbations is of fundamental interest and could identify new targets for antibiotic development.The initial interaction between bacteria and the host is mediated by micrometer-long adhesive structures termed pili or fimbriae (Fig. 1A). Due to their adhesive role, pili are virulence factors that contribute to the development of infections (6). Structurally, pili are polymers of tens to hundreds of subunits, termed shaft pilins, that are assembled in series and are presented at the extracellular surface, often with inclusion of minor pilins that can have adhesive properties (6). Remar...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

Echelman

Alegre-Cebollada

Badilla

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Using this approach, we could avoid the formation of high-molecular-weight (HMW) glycosylated GspA by forcing the protein to be released as a monomer to facilitate the detection of small changes in preprotein processing. After harvesting of the cell-free culture supernatants of A. oris strains expressing this construct by centrifugation and filtration, His-tagged proteins were captured by Ni-nitrilotriacetic acid agarose and purified according to a previously published protocol (7). Purified proteins were analyzed by SDS-PAGE and then Coomassie blue and periodic acid-Schiff (PAS) staining (see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

“…Type 2 fimbriae-composed of the shaft fimbrillin FimA and the tip fimbrillin FimB-promote polymicrobial interactions or bacterial coaggregation, biofilm formation, and adhesion to host cells (2,5,6). A. oris also encodes 14 cell wall-anchored proteins (AcaA to AcaN), one of which, CafA (previously named AcaF), has been shown to be the major coaggregation factor that forms a distinct fimbrial tip of type 2 fimbriae (7). It was recently shown that the cell wall-anchored protein AcaC (named GspA) is glycosylated, and its glycosylation appears to be coupled to protein secretion and sortase SrtA-catalyzed cell wall anchoring (8).…”

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confidence: 99%

A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris

Siegel

Ton‐That

2016

J Bacteriol

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The Gram-positive bacterium Actinomyces oris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motifcontaining proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria, A. oris expresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal "glyco-stress," caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortase srtA is genetically disrupted. Consistent with this finding, we show here that a mutant lacking lepB2 and srtA was unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neither lepB1 nor lepB2 abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, the lepB2 deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, the lepB2 mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely, lepB1 was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity. A ctinomyces oris is a key colonizer in the development of dental plaque or oral biofilms because of its ability to adhere to many oral colonizers and host surfaces. This multiadhesive property is attributed to the presence of two types of fimbriae or pili called type 1 and 2 fimbriae (1, 2). Type 1 fimbriae-made of the shaft fimbrillin FimP and the tip fimbrillin FimQ-mediate bacterial adherence to proline-rich proteins that coat tooth enamel (2-4). Type 2 fimbriae-composed of the shaft fimbrillin FimA and the tip fimbrillin FimB-promote polymicrobial interactions or bacterial coaggregation, biofilm formation, and adhesion to host cells (2,5,6). A. oris also encodes 14 cell wall-anchored proteins (AcaA to AcaN), one of which, CafA (previously named AcaF), has been shown to be the major coaggregation factor that forms a distinct fimbrial tip of type 2 fimbriae (7). It was recently shown that the cell wall-anchored protein AcaC (named GspA) is glycosylated, and its glycosylation appears to be coupled to protein secretion and sortase SrtA-catalyzed cell wall anchoring (8). Shared features found in these cell wall-anchored proteins and pilins (or fimbrillins) are an N-terminal signal peptide and a C-terminal cell wall sorting signal (CWSS). The N-terminal signal...

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Bacterial Pili and Fimbriae

Ramirez

Ton‐That

2020

Encyclopedia of Life Sciences

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Bacterial proteinaceous filaments termed pili or fimbriae are nonflagellar, hair‐like structures protruding from the cell surface that are critical for bacterial virulence and fitness. Present in both Gram‐negative and Gram‐positive bacteria, pili are involved in many processes such as conjugation, adherence, twitching motility, biofilm formation and immunomodulation. Considerably diverse and complex, Gram‐negative pili are formed by noncovalent polymerisation of various pilin subunits; many of these pili require chaperones and usher proteins for their assembly. In contrast, fewer pilus systems have been described for the Gram‐positive counterparts; notably well studied are the heterotrimeric or ‐dimeric pili that are covalently assembled by a transpeptidase enzyme called sortase. Furthermore, type IV pili have been identified in several Gram‐positive bacteria, especially in clostridia. Key Concepts Noncovalent pilus polymerisation is a general mechanism of pilus assembly in Gram‐negative bacteria, including the chaperone/usher assembly pathway and type IV pilus pathway, which generates noncovalent pilus polymers. Sortase‐mediated pilus assembly is a general mechanism of pilus assembly in Gram‐positive bacteria that involves a transpeptidase enzyme named sortase, which cleaves pilin precursors at sorting signals between threonine and glycine residues and utilises the side‐chain amino groups of pilin motif sequences to generate covalent links between pilin subunits. The sorting signal consists of an LPXTG motif followed by a hydrophobic domain and a positively charged tail. The pilin motif is an 11 amino acid sequence of WxxxVxVYPKN located near the N ‐terminus of major pilin subunits, in which the electron‐donating lysine residue forms an isopeptide bond with the threonine residue generated from the cleavage of the LPXTG motif of adjacent pilin subunits. Tissue tropism is referred to as the ability of a pathogen to adhere specifically to some particular epithelial cells. Phase variation involves switching of surface antigens such as pili that allows a pathogen to evade the host immune system. Immunomodulation is a process of changing the host's immune system by certain molecules known as immunomodulators including pili that can activate or suppress immune cells. Dental plaque is one of the most complex bacterial biofilms that is formed by sequential colonisation of initial colonisers such as Actinomyces spp. and oral streptococci and late colonisers; this process involves Actinomyces fimbriae. Twitching motility mediated by pili allows translocation between mucosal surfaces, colonisation of host tissues and establishment of biofilms by a pathogen.

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Pilus hijacking by a bacterial coaggregation factor critical for oral biofilm development

Cited by 49 publications

References 26 publications

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomyces oris

Bacterial Pili and Fimbriae

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