The ability to adhere to intestinal epithelial tissue and mucosal surfaces is a key criterion in selecting probiotics. Adhesion is considered to be a prerequisite for successful colonization and survival in the gastrointestinal tract to provide persistent beneficial effects to the host. Bacteria express a multitude of surface components that mediate adherence. Pili or fimbriae are surface adhesive components implicated in initiating bacterial adhesion and mediating interaction with the host. These nonflagellar proteinaceous fiber appendages were identified and explored over several decades in pathogenic bacteria, and many distinct types are known. However, the presence of pili in probiotics and/or commensalic bacteria has only recently been recognized. Thus knowledge about pili in probiotics is relatively limited, but structural and functional data have begun to emerge. Availability of these data in the future would enable us to understand the pili-mediated adhesion strategies of probiotics. This knowledge could be utilized to develop antiadhesion-based therapies against bacterial infections as well as probiotic designs for beneficial effects. This chapter will briefly summarize the current knowledge of pili in probiotics with emphasis on members of lactobacilli and bifidobacteria.
Bacterial colonization of humans seems to commence at birth and evolves throughout life. It depends on several factors including mode of birth, age, geographical location, local environment, diet, stress, illness, medications, and antibiotic treatment. Bacteria colonize all parts of the human body that are exposed to external environment. Specifically, the gastrointestinal tract (GIT) harbors more than 1000 species, and this complex microbial community is referred to as the “gut microbiota” [1, 2]. The gut microbiota are well recognized because of their impact on health and disease. However, knowledge on the precise mechanism(s) by which the microbiota exerts its influence remains largely unknown.
Most pathogenic bacteria are known to express multitude of surface components for establishing contacts and mediating interactions with the host for bacterial colonization. Among these, long, hair‐like filamentous structures known as pili or fimbriae have been often implicated in adhesion processes and shown to be required for bacterial colonization on host tissues (for reviews, see [5–11]). Typically, these structures are made up of building blocks called pilins or fimbrilins. Genes for these pilins along with other genes required for the pilus assembly are located in the same place in the genome called pilus gene cluster or Pathogenicity Island. Distinct pilus structures (e.g., chaperone‐mediated, type IV, Curli, and CS1) are known in Gram‐negative pathogens. Their structure, function, and biogenesis have been well explored to some extent. The details of pili have begun to emerge for Gram‐positive pathogens a decade ago (for reviews, see [8, 10–15]). The sortase‐mediated pili seem to be conserved across the Gram‐positive pathogens. Some of the pilus types (e.g., type IV) exist both in the Gram‐negative and Gram‐positive pathogens. The pilus types have been majorly categorized based on secretion systems, biogenesis, architecture, and function. The sortase‐mediated pili differ from other known types by being a covalent polymer in which pilin subunits are covalently tethered to each other by sortase‐mediated isopeptide bonds. The pili and their components in the pathogens are recognized as virulence factors as they play a key role in pathogenesis. Also, they are considered as potential vaccine candidates because of their immunogenic properties.
Although the focus is traditionally on pili in pathogenic bacteria for last few decades, they have been recently identified in many gut commensalic bacteria and often shown to be essential for their colonization and persistence in the the GIT and for immune modulation. Although the pili in pathogenic bacteria are regularly reviewed, this chapter attempts to give a brief overview of pili in beneficial bacteria, which is relatively recent.
2. Sortase‐mediated pili
As demonstrated first in pathogen
The pilins are commonly made up of two building blocks, which are variants of immunoglobulin fold known as CnaA  and CnaB , often with intradomain isopeptide bond  (for reviews, see [20–22]) (Figure 2). In addition, the tip pilins also contain adhesin modules such as von Willebrand factor type A domain (vWFA) with two inserted arms [23, 24] and thioester containing domains [25–27] (Figure 2). The pilus model of
The sortase‐mediated pili, which are being actively investigated in Gram‐positive pathogens and considered as virulence factors, have been detected in several gut commensals as mentioned in the following sections. The pilus‐like gene clusters were earlier noticed in probiotic
2.1. Pili in
L. rhamnosus GG
Obtaining three‐dimensional structural insights into pilus assembly and adhesion mechanisms through the structural biology techniques has been instrumental for Gram‐negative pathogens in the past (for reviews, see [5, 8, 49, 50]), and it was begun much later for Gram‐positive pathogens in 2007 ([19, 51], for reviews, see [11, 20–22]). The structures of individual major as well as ancillary pilins from several pathogenic strains have been determined (for recent review, see ) (Figure 2). A Cryo‐EM study on
Detailed structural knowledge is yet to emerge for pili and related components for probiotic bacteria. However, preliminary crystallographic data are available for some of the pilins (SpaA , SpaD , and SpaC ) in
2.2. Pili in
2.3. Pili in other
The presence of sortase‐mediated pilus gene clusters has been reported in many strains of
2.4. Pili in
2.5. Pili in bifidobacteria
Bifidobacteria are the common components of the gut microbiota of a broad range of hosts . Several members of bifidobacteria are typical inhabitants of the infant intestine , which is thought to be sterile at birth. Identification of many bifidobacterial strains in the stools of healthy infants suggests that they could be the first colonizers in the GIT subsequent to birth. Genomic analysis has revealed pilus genes cluster in several bifidobacterial strains . Interestingly, many pilus gene clusters are flanked by transposon elements indicating their acquisition by HGT. The presence of pilus structures was further examined by AFM and transcription analysis in
3. Tad pili
The Tad (tight adherence) pili, which was first described in
Type IV pilus assembly is a complex process, which requires protein products from multiple genes (∼14) including minor pilins, prepilin peptidase, ATPase, inner membrane core proteins, and accessory proteins. Many of the core genes are conserved across different bacterial species. Tad pili seem to differ from other type IV pilus types by lacking four core homologous minor pilins. The type IV pilins are synthesized as precursors with a leader peptide and transported across the inner membrane into the periplasmic space, where they are retained in the inner membrane through their N‐terminal hydrophobic segments. The globular domain is folded with stabilizing intramolecular disulfide bonds. A dedicated prepilin peptidase cleaves the positively charged leader sequence and methylates the N‐terminal amine to generate the mature pilin. The methylated, positively charged N‐terminal residue is thought to attract negatively charged glutamate (at fifth position) of adjacent major pilin in the growing pilus fiber. This results in vertical displacement between one pilin and the next. The assembly ATPase associated with the cytoplasmic part of the inner membrane protein undergoes conformational change during ATP hydrolysis and pushes the pilus filament out of the membrane, providing a gap for the next major pilin. Type IV pili is further complicated by divergence and divided into two classes (types IVa and IVb) based on the length of leader peptides and mature pilins. The pilins of type IVa are typically 150‒160 residues long with a short leader peptide (<10 residues), whereas the pilins of type IVb are either long (180‒200 residues) or short (40‒50 residues) with longer leader peptides (∼15‒30 residues). The Tad pili are monophyletic subclass of type IVb pili . The pilins of Tad pili are short with 40‒50 residues long.
Though the sequence and structural diversity are associated with the pilins in type IV, they share a common lollipop‐like architecture consisting of an extended N‐terminal helical stick followed by a globular head containing a β‐sheet with 4–7 strands  (Figure 4B). The N‐terminal half of the helix is hydrophobic and multifunctional regulatory domain. It protrudes from the globular head and forms the central hydrophobic core of the growing filament during the pilus assembly. Prior to assembly, it acts as transmembrane segment to retain individual pilin in the cytoplasmic membrane. The C‐terminal half of the helix is amphipathic and embedded in the globular head. For many pili, a hypervariable C‐terminal loop known as D‐region or disulfide‐bonded loop (DSL) performs an essential role in surface adherence (Figure 4B). The conserved disulfide bridge in the D‐region observed in several Gram‐negative major pilins appears to be off in Gram‐positive pilins (e.g., PilA1 in
3.1. Tad pili in
Apart from sortase‐dependent pili,
4. Future perspectives
Adhesion of bacteria to host surfaces is a prerequisite and crucial step for bacterial colonization, which may result in pathogenic or commensal relationship. The pili have been often implicated in initiating adhesion and mediating interaction with host. Understanding pilus structure and function, and their mediated interactions with the host has been achieved to a certain extent in pathogenic strains. The pili and their components are recognized as virulence factors in pathogenic strains, and also considered as potential vaccine candidates in combating bacterial infection. Recent identification of such surface organelles in probiotic or commensal bacteria gives a new perspective as a niche‐adaption factor as well. The sortase‐mediated pili initially discovered in Gram‐positive pathogens appear to be widespread among commensals. The Tad pili, which are known to present in both Gram‐negative and Gram‐positive pathogens, have also been detected in some commensal strains. It may not be a surprise if additional pilus type comes in the future from the fast‐growing technology and genomes for gut microbiota. Available preliminary data suggest that the pili from pathogenic and beneficial bacteria share several sequence and structural features. The presence of transposable element in several pilus gene clusters indicates that the pathogenic and commensal bacteria may be acquired from each other during the evolution. The challenge is now to understand the differences between the (enemy) pathogenic and (friendly) beneficial bacteria in their pili‐mediated adhesion strategies and interactions with the host. This knowledge is crucial in optimizing probiotics and targeting adhesion‐based therapies for human health. The journey of pilus research in probiotics has begun with the prototype SpaCBA pili in
This work was funded by the Regional Centre for Biotechnology (RCB) and the Department of Biotechnology (DBT) (Grant No. BT/PR5891/BRB/10/1098/2012), India.
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