New Paradigms of Pilus Assembly Mechanisms in Gram-Positive Actinobacteria

doi:10.1016/j.tim.2020.05.008

Trends in Microbiology

Volume 28, Issue 12, December 2020, Pages 999-1009

https://doi.org/10.1016/j.tim.2020.05.008 Get rights and content

Highlights

Covalently linked pili are assembled on the cell surface of many Gram-positive bacteria via a biphasic mechanism whereby pilus polymerization is catalyzed by the pilus-specific sortase followed by cell wall anchoring of pili by the housekeeping sortase.
Pilus-mediated adhesion depends on pilus length, which is modulated by the housekeeping sortase via unique structural features.
Some Gram-positive surface proteins with the LPXTG motif may hijack a pilus assembly machine via molecular mimicry to be displayed at the pilus tip.
Pilus-specific sortase enzymes provide a bioconjugation tool via the formation of an isopeptide bond that is mechanically stable and less susceptible to proteolytic cleavage.

Adhesive pili in Gram-positive bacteria represent a variety of extracellular multiprotein polymers that mediate bacterial colonization of specific host tissues and associated pathogenesis. Pili are assembled in two distinct but coupled steps, an orderly crosslinking of pilin monomers and subsequent anchoring of the polymer to peptidoglycan, catalyzed by two transpeptidase enzymes – the pilus-specific sortase and the housekeeping sortase. Here, we review this biphasic assembly mechanism based on studies of two prototypical models, the heterotrimeric pili in Corynebacterium diphtheriae and the heterodimeric pili in Actinomyces oris, highlighting some newly emerged basic paradigms. The disparate mechanisms of protein ligation mediated by the pilus-specific sortase and the spatial positioning of adhesive pili on the cell surface modulated by the housekeeping sortase are among the notable highlights.

Section snippets

Covalently-Linked Pili of Gram-Positive Bacteria

Fiber-like appendages called ‘pili’ or ‘fimbriae’ are microscopic structures present on the cell surface of both Gram-negative and Gram-positive bacteria. They are involved in a wide range of cellular activities, including adherence, motility, conjugation, and virulence [1., 2., 3.]. Among these, the only pilus form known to date in which individual subunits are covalently bonded is the pili that are assembled by the action of sortase enzymes conserved in Gram-positive bacteria [4], but not in

Assembly of the SpaA Pilus

The causative agent of human diphtheria C. diphtheriae is among the earliest bacterial species where pili were identified [22,23]. As in many Gram-positive bacteria [4], the C. diphtheriae genes coding for distinct pilin subunits and dedicated pilus-specific sortases, which are all class C sortases, are organized into three operons [10]. Together, they encode three distinct pilus types specified by their major subunits: SpaA-type, SpaD-type, and SpaH-type pili [10,24,25]. Pili constitute one of

Heterodimeric Fimbriae of Actinomyces oris

Actinomyces are one of the most dominant and earliest colonizing genera of microbes present in the human oral cavity, with A. oris (formerly called Actinomyces naeslundii) detected in children as young as 1 year old [42,43]. A. oris is a major contributor to dental plaque through its ability to coaggregate with other microbial species and thus a key to the genesis of complex biofilms on the surface of teeth and the mucosal epithelia [44., 45., 46.]. This intrinsic adherence property of A. oris

Concluding Remarks

Collective efforts during the last decade dissected the molecular assembly mechanisms of Gram-positive pili and probed their roles in bacterial pathogenesis and their use in the development of vaccines. While pilus vaccines have yet to emerge in the clinical arena, we now have made great strides in the basic biology and have a clearer view of pilus biogenesis in Gram-positive bacteria. A common feature in these monoderms is the biphasic mode of pilus assembly by distinct steps of enzymatic

Acknowledgements

We would like to dedicate this review to our late former mentor and colleague Dr Olaf Schneewind, who discovered the cell wall anchoring pathway of Gram-positive bacterial surface proteins. His amazing insights and warm friendship will be sorely missed. We thank our laboratory members for critical review of the manuscript and for discussion. Work in the Ton-That laboratory related to Gram-positive pili has been supported by the National Institute of Dental and Craniofacial Research

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Dental plaque is a complex microbial biofilm community of many species and a major cause of oral infections and infectious endocarditis. Plaque development begins when primary colonizers attach to oral tissues and undergo coaggregation. Primary colonizers facilitate cellular attachment and inter-bacterial interactions through sortase-dependent pili (or fimbriae) extending out from their cell surface. Consequently, the sortase enzyme is viewed as a potential drug target for controlling biofilm formation and avoiding infection. Streptococcus sanguinis is a primary colonizing bacterium whose pili consist of three different pilin subunits that are assembled together by the pilus-specific (C-type) SsaSrtC sortase. Here, we report on the crystal structure determination of the recombinant wild-type and active-site mutant forms of SsaSrtC. Interestingly, the SsaSrtC structure exhibits an open-lid conformation, although a conserved DPX motif is lacking in the lid. Based on molecular docking and structural analysis, we identified the substrate-binding residues essential for pilin recognition and pilus assembly. We also demonstrated that while recombinant SsaSrtC is enzymatically active toward the five-residue LPNTG sorting motif peptide of the pilins, this activity is significantly reduced by the presence of zinc. We further showed that rutin and α-crocin are potential candidate inhibitors of the SsaSrtC sortase via structure-based virtual screening and inhibition assays. The structural knowledge gained from our study will provide the means to develop new approaches that target pilus-mediated attachment, thereby preventing oral biofilm growth and infection.
Bacterial pili and fimbriae
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This chapter covers our current understanding of diverse pili discovered and characterized in gram-negative bacteria. We focus on three main pili, type I and P pilus, type IV pilus, and type V pilus. We compare and contrast their assembly mechanisms, discuss their biological activities and virulent potentials, outline their environmental, genetic and biochemical regulations, and highlight their potential to become novel therapeutic targets to develop precision antiinfective therapies that circumvent emerging antimicrobial resistance and fight an epidemic of antibiotic-resistant strains in postantibiotic era.
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Nocardiosis is an opportunistic infectious disease caused by a ubiquitous aerobic bacterium of the genus Nocardia that can be found in the environment. These bacteria are held responsible for many infections affecting the lungs, the brain or the skin—especially in immunocompromised patients. The taxonomy of this bacterium is complex, and a multilocus sequence analysis may be sometimes necessary for a correct identification when the 16S rRNA gene does not yield discriminative enough results. Nowadays, the matrix-assisted laser desorption ionization time-of-flight mass spectrometry system offers an interesting possibility to this genus as it allows to rapidly identify with a low error rate the four most common clinical species or species complexes: Nocardia farcinica, Nocardia abscessus, Nocardia nova, and Nocardia cyriacigeorgica. The next-generation sequencing system allows researchers to easily obtain genomic sequences at an affordable cost, thereby facilitating taxonomic analyses and even enhancing the accuracy of identifications. Actinomyces belong to the normal indigenous microflora, therefore they are considered as facultative pathogens. Actinomycosis is usually associated with the breakdown of normal physical barriers, such as disruption of mucosal membranes. Actinomycosis and nocardiosis are distinct diseases that respond to very different forms of therapy. Actinomyces can be readily distinguished from Nocardia by their different—anaerobic versus aerobic—patterns of growth after isolation from a clinical sample.
A review on pilus assembly mechanisms in Gram-positive and Gram-negative bacteria
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The complete assembly mechanism of pili subunits and their functions are yet to be studied. Collective studies and reviews on the pilus assembly mechanism of Gram-positive and Gram-negative bacteria and their role in bacterial species created a path for drug and vaccine development (Govindarajan et al., 2020; Kline et al., 2010; Ramirez et al., 2020). This article aims to provide a clear and concise review of the pilus assembly mechanism by elaborating on the role of individual pilin subunits along with their physiological functions.
The surface of Gram-positive and Gram-negative bacteria contains long hair-like proteinaceous protrusion known as pili or fimbriae. Historically, pilin proteins were considered to play a major role in the transfer of genetic material during bacterial conjugation. Recent findings however elucidate their importance in virulence, biofilm formation, phage transduction, and motility. Therefore, it is crucial to gain mechanistic insights on the subcellular assembly of pili and the localization patterns of their subunit proteins (major and minor pilins) that aid the macromolecular pilus assembly at the bacterial surface. In this article, we review the current knowledge of pilus assembly mechanisms in a wide range of Gram-positive and Gram-negative bacteria, including subcellular localization patterns of a few pilin subunit proteins and their role in virulence and pathogenesis.
Length control of long cell protrusions: Rulers, timers and transport
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Subsequently the pili were found also on the surface of Gram-positive bacteria. One distinction between the pili of Gram-positive and Gram-negative bacteria is that the monomeric subunit are bonded covalently in the former but non-covalently in the latter [31]. Pili or fimbriae are involved in wide range of functions including motility as well as adhesion to other cells and environmental surfaces [252].
Living cells use long tubular appendages for locomotion and sensory purposes. Hence, assembling and maintaining a protrusion of correct length is crucial for survival and overall performance. Usually the protrusions lack the machinery for the synthesis of building blocks and imports them from the cell body. What are the unique features of the transport logistics which facilitate the exchange of these building blocks between the cell and the protrusion? What kind of ‘rulers’ and ‘timers’ does the cell use for constructing its appendages of correct length on time? How do the multiple appendages coordinate and communicate among themselves during different stages of their existence? How frequently do the fluctuations drive the length of these dynamic protrusions out of the acceptable bounds? These questions are addressed from a broad perspective in this review which is organized in three parts. In part-I the list of all known cell protrusions is followed by a comprehensive list of the mechanisms of length control of cell protrusions reported in the literature. We review not only the dynamics of the genesis of the protrusions, but also their resorption and regrowth as well as regeneration after amputation. As a case study in part-II, the specific cell protrusion that has been discussed in detail is eukaryotic flagellum (also known as cilium); this choice was dictated by the fact that flagellar length control mechanisms have been studied most extensively over more than half a century in cells with two or more flagella. Although limited in scope, brief discussions on a few non-flagellar cell protrusions in part-III of this review is intended to provide a glimpse of the uncharted territories and challenging frontiers of research on subcellular length control phenomena that awaits rigorous investigations.
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Surface pili (or fimbriae) are an important but conspicuous adaptation of several genera and species of Gram-negative and Gram-positive bacteria. These long and non-flagellar multi-subunit adhesins mediate the initial contact that a bacterium has with a host or environment, and thus have come to be regarded as a key colonization factor for virulence activity in pathogens or niche adaptation in commensals. Pili in pathogenic bacteria are well recognized for their roles in the adhesion to host cells, colonization of tissues, and establishment of infection. As an ‘anti-adhesive’ ploy, targeting pilus-mediated attachment for disruption has become a potentially effective alternative to using antibiotics. In this review, we give a description of the several structurally distinct bacterial pilus types thus far characterized, and as well offer details about the intricacy of their individual structure, assembly, and function. With a molecular understanding of pilus biogenesis and pilus-mediated host interactions also provided, we go on to describe some of the emerging new approaches and compounds that have been recently developed to prevent the adhesion, colonization, and infection of piliated bacterial pathogens.

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Trends in Microbiology

ReviewNew Paradigms of Pilus Assembly Mechanisms in Gram-Positive Actinobacteria

Highlights

Section snippets

Covalently-Linked Pili of Gram-Positive Bacteria

Assembly of the SpaA Pilus

Heterodimeric Fimbriae of Actinomyces oris

Concluding Remarks

Acknowledgements

Trends Microbiol.

Trends Microbiol.

Trends Microbiol.

Res. Microbiol.

Curr. Opin. Microbiol.

Res. Microbiol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

Surface organelles assembled by secretion systems of Gram-negative bacteria: diversity in structure and function

FEMS Microbiol. Rev.

Sortase enzymes in Gram-positive bacteria

Mol. Microbiol.

Identification of a gene involved in assembly of Actinomyces naeslundii T14V type 2 fimbriae

Infect. Immun.

Synthesis and function of Actinomyces naeslundii T14V type 1 fimbriae require the expression of additional fimbria-associated genes

Infect. Immun.

Assembly of pili on the surface of Corynebacterium diphtheriae

Mol. Microbiol.

Sortases and pilin elements involved in pilus assembly of Corynebacterium diphtheriae

Mol. Microbiol.

Assembly of pili on the surface of Bacillus cereus vegetative cells

Mol. Microbiol.

Endocarditis and biofilm-associated pili of Enterococcus faecalis

J. Clin. Invest.

Genome analysis reveals pili in group B Streptococcus

Science

Assembly and role of pili in group B streptococci

Mol. Microbiol.

Streptococcus pyogenes pili promote pharyngeal cell adhesion and biofilm formation

Mol. Microbiol.

Sortase-mediated assembly and surface topology of adhesive pneumococcal pili

Mol. Microbiol.

Role of sortase-dependent pili of Bifidobacterium bifidum PRL2010 in modulating bacterium-host interactions

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

Pili in Gram-positive pathogens

Nat. Rev. Microbiol.

Architects at the bacterial surface – sortases and the assembly of pili with isopeptide bonds

Nat. Rev. Microbiol.

Adhesion by pathogenic corynebacteria

Adv. Exp. Med. Biol.

Presence of pili in species of human and animal parasites and pathogens of the genus Corynebacterium

Infect. Immun.

Assembly of distinct pilus structures on the surface of Corynebacterium diphtheriae

J. Bacteriol.

Type III pilus of corynebacteria: pilus length is determined by the level of its major pilin subunit

J. Bacteriol.

A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence

Mol. Microbiol.

Housekeeping sortase facilitates the cell wall anchoring of pilus polymers in Corynebacterium diphtheriae

Mol. Microbiol.

Sortase A utilizes an ancillary protein anchor for efficient cell wall anchoring of pili in Streptococcus agalactiae

Infect. Immun.

Pilin and sortase residues critical for endocarditis- and biofilm-associated pilus biogenesis in Enterococcus faecalis

J. Bacteriol.

Contribution of individual Ebp pilus subunits of Enterococcus faecalis OG1RF to pilus biogenesis, biofilm formation and urinary tract infection

PLoS One

The molecular switch that activates the cell wall anchoring step of pilus assembly in gram-positive bacteria

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

In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

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

Review
New Paradigms of Pilus Assembly Mechanisms in Gram-Positive Actinobacteria