Crystal structure of the pilus-specific sortase from early colonizing oral Streptococcus sanguinis captures an active open-lid conformation.

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.

Deciphering the substrate specificity of housekeeping sortase A and pilus-specific sortase C of probiotic bacterium Lactococcus lactis.

Several strains and species of lactic acid bacteria (LAB) are widely used in fermented foods, including dairy products and also as probiotics, because of their contribution to various health benefits in humans. Sortase enzymes decorate the bacterial cell wall with different surface proteins and pili for facilitating the interactions with host and environment for the colonization and beneficial effects. While the sortases and sortase anchored proteins from pathogens have been the prime focus of the research in the past, sortases from many non-pathogenic bacteria, including LAB strains, have attracted attention for their potential applications in vaccine delivery and other clinical interventions. Here, we report the purification and functional characterization of two sortases (housekeeping SrtA and pilus-specific SrtC) from a probiotic Lactococcus lactis. The purified sortases were found to be active against the putative LPXTG motif-based peptide substrates, albeit with differences. The in-silico analysis provides insights into the residues involved in substrate binding and specificity. Overall, this study sheds new light on the aspects of structure, substrate specificity, and function of sortases from non-pathogenic bacteria, which may have physiological ramifications as well as their applications in sortase-mediated protein bioconjugation.

New structural insights into the PI-2 pilus from Streptococcus oralis, an early dental plaque colonizer.

Streptococcus oralis is a member of the mitis group of oral streptococci and an early colonizer in dental plaque biofilm, a major cause of periodontal disease, dental caries, and other oral infections. S. oralis promotes biofilm growth by coaggregating in a mutualistic partnership with other early colonizers such as Actinomyces oris. For this cell-to-cell interaction, A. oris is known to use its sortase-dependent pilus (type 2), but whether S. oralis uses its PI-2 (pilus islet 2) pilus is still to be determined. The PI-2 pilus is predicted to have a heterodimeric structure consisting of two different protein subunits with their own location and function: the tip PitA pilin for adhesion and the backbone PitB pilin for length. Thus far, structural information remains incomplete about the role of PI-2 pili in the mutualistic mechanism between S. oralis and A. oris. We now report on the crystal structure analysis of PitA and PitB using X-ray crystallography, small-angle X-ray scattering, and molecular docking studies. Accordingly, we propose a structural model for the PI-2 pilus, wherein repeating PitB subunits are arranged head-to-tail to form the long backbone structure with PitA on the outer tip. By performing both in vitro and in vivo experiments, we examined the role played by PitA in mediating the mutualistic interaction between S. oralis and A. oris, which appears to involve the coaggregation factor CafA. We also reveal that the galactose monosaccharide is a conceivable ligand for PitA and thereby might be used to inhibit coaggregation and control oral biofilm development. DATABASE: Structural coordinates for the PitA fragment, PitA fragment TbXO4 derivative, full-length PitA, and PitB from S. oralis have been deposited at the Protein Data Bank as 7VCR, 7W7I, 7VCN, 7W6B, and 7W7I, respectively. Streptococcus pneumoniae PitB coordinates have been deposited as 7F7Y.

Wheatgrass inhibits the lipopolysaccharide-stimulated inflammatory effect in RAW 264.7 macrophages.

Inflammation is a multifaceted set of cellular communications generated against foreign infection, toxic influence or autoimmune injury. The present study investigates the anti-inflammatory effect of wheatgrass extract against the harmful impact of lipopolysaccharide (LPS) in macrophage cells, i.e., RAW 264.7 cells. Our results indicate that 5- and 7- days old wheatgrass extracts inhibit the LPS-stimulated production of nitric oxide. Moreover, wheatgrass extract significantly downregulates the mRNA expression of LPS-stimulated various pro-inflammatory markers, tumor necrosis factor-α, interleukin-6, interleukin-1ß, AP-1 and also iNOS-2 and COX-2. Our flow cytometry analyses confirmed that wheatgrass extract prevents the generation of reactive oxygen species in LPS-stimulated RAW 264.7 cells, thus arresting oxidative stress in cells. The immunoblot analyses also confirmed a significant reduction in the expression of inflammatory proteins, namely, iNOS-2 and COX-2, in wheatgrass extract-treated cells, compared to LPS-stimulated condition. The NF-κB transactivation assay further confirmed the inhibitory effect of wheatgrass extracts on the LPS-stimulated expression of NF-κB. Molecular docking based studies showed the plausible binding of two significant wheatgrass constituents, i.e., apigenin and myo-inositol with COX-2 protein, with binding energies of -10.59 kcal/mol and -7.88 kcal/mol, respectively. Based on the above results, wheatgrass may be considered as a potential therapeutic candidate for preventing inflammation.

LrpCBA pilus proteins of gut-dwelling Ligilactobacillus ruminis: crystallization and X-ray diffraction analysis.

Adhesion to host surfaces for bacterial survival and colonization involves a variety of molecular mechanisms. Ligilactobacillus ruminis, a strict anaerobe and gut autochthonous (indigenous) commensal, relies on sortase-dependent pili (LrpCBA) for adherence to the intestinal inner walls, thereby withstanding luminal content flow. Here, the LrpCBA pilus is a promiscuous binder to gut collagen, fibronectin and epithelial cells. Structurally, the LrpCBA pilus displays a representative hetero-oligomeric arrangement and consists of three types of pilin subunit, each with its own location and function, i.e. tip LrpC for adhesion, basal LrpB for anchoring and backbone LrpA for length. To provide further structural insights into the assembly, anchoring and functional mechanisms of sortase-dependent pili, each of the L. ruminis pilus proteins was produced recombinantly for crystallization and X-ray diffraction analysis. Crystals of LrpC, LrpB, LrpA and truncated LrpA generated by limited proteolysis were obtained and diffracted to resolutions of 3.0, 1.5, 2.2 and 1.4 Å, respectively. Anomalous data were also collected from crystals of selenomethionine-substituted LrpC and an iodide derivative of truncated LrpA. Successful strategies for protein production, crystallization and derivatization are reported.

Exploiting pilus-mediated bacteria-host interactions for health benefits.

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.

Black pepper and piperine induce anticancer effects on leukemia cell line.

The black pepper, most commonly used in Indian cuisines for ages, is considered as "king of spices." The present study evaluates the anticancer potential of black pepper and its main constituent, i.e. alkaloid piperine, against human leukemia cell line, K-562 cells. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed the presence of piperine in black pepper extract. The methanolic extract of black pepper (BP-M) and pure piperine (PIP) showed a strong cytotoxic effect against this cell line. Both BP-M and PIP generated apoptotic bodies in K-562 cells and caused nuclear condensation as visualized by fluorescent microscopy, which was further confirmed by flow cytometry analysis. BP-M and PIP also generated reactive oxygen species in K-562 cells as established by flow cytometry. The translation of Bax, caspase-3 and caspase-9 genes was found to be upregulated with subsequent downregulation of Bcl-2 gene. The anti-proliferative effect of both BP-M and PIP was also observed by trypan blue staining and was further confirmed by the downregulated expression of proliferating cell nuclear antigen (PCNA). The molecular docking studies showed the binding of PIP with PCNA and Bcl-2 and supported the in vitro findings. The docking studies also proposed the binding of PIP to ADP binding pocket of Apaf-1 protein. Taken together, these findings signify the anticancer potential of both black pepper and PIP, thus proposing black pepper as a potent nutraceutical for preventing the progression of chronic myeloid leukemia.

Black pepper prevents anemia of inflammation by inhibiting hepcidin over-expression through BMP6-SMAD1/ IL6-STAT3 signaling pathway.

Hepcidin, a circulatory hepatic peptide hormone, is associated with systemic iron homeostasis. Inflammation leads to an increase in hepcidin expression, which dysregulates body iron level. The related disorder, anemia of inflammation, is the second most prevalent anemia-related disorder worldwide. In the present study, we conducted in vitro and in vivo studies to evaluate the effect of black pepper (BP) and its major bioactive alkaloid, piperine, on anemia of inflammation. The initial in vitro study using human hepatocyte cell line, HepG2, confirmed that among different black pepper extracts: methanol (BPME), ethanol (BPEE) and aqueous (BPAE), BPME to be most effective in downregulating transcription of hepcidin gene. Further, BPME and piperine significantly downregulated hepcidin protein expression at 200 µg/ml and 100 µM concentrations, respectively. In the next phase, BPME and piperine were found to significantly attenuate BMP-6 and IL-6 induced hepcidin overexpression by downregulating the increased level of pSMAD1 and pSTAT3 proteins, respectively. For in vivo study, we first subcutaneously injected male BALB/c mice with oil of turpentine, thrice within a period of two weeks, in order to enhance the expression of hepcidin. After that, the intraperitoneal administration of BPME and piperine at 70 and 25 mg/kg body weight, respectively, on alternate days for a period of another two weeks resulted in downregulation of hepcidin overexpression in diseased mice, as confirmed by RT-PCR and immunoblot analysis. The histopathology of liver tissue confirmed increased iron bioavailability in BPME and piperine treated animals. The molecular docking-based interaction studies demonstrated the binding potential of piperine with SMAD1 and STAT3 proteins. The binding patterns supported the proposed inhibition of hepcidin activating proteins. All together, these findings suggest black pepper as a therapeutic candidate for the treatment of anemia of inflammation.

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

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.

The adhesive PitA pilus protein from the early dental plaque colonizer Streptococcus oralis: expression, purification, crystallization and X-ray diffraction analysis.

PitA is the putative tip adhesin of the pilus islet 2 (PI-2)-encoded sortase-dependent pilus in the Gram-positive Streptococcus oralis, an opportunistic pathogen that often flourishes within the diseased human oral cavity. Early colonization by S. oralis and its interaction with Actinomyces oris seeds the development of oral biofilm or dental plaque. Here, the PI-2 pilus plays a vital role in mediating adherence to host surfaces and other bacteria. A recombinant form of the PitA adhesin has now been produced and crystallized. Owing to the large size (∼100 kDa), flexibility and complicated folding of PitA, obtaining diffraction-quality crystals has been a challenge. However, by the use of limited proteolysis with α-chymotrypsin, the diffraction quality of the PitA crystals was considerably enhanced to 2.16 Šresolution. These crystals belonged to space group P1, with unit-cell parameters a = 61.48, b = 70.87, c = 82.46 Å, α = 80.08, ß = 87.02, γ = 87.70°. The anomalous signal from the terbium derivative of α-chymotrypsin-treated PitA crystals prepared with terbium crystallophore (Tb-Xo4) was sufficient to obtain an interpretable electron-density map via terbium SAD phasing.

Crystal structure of the atypically adhesive SpaB basal pilus subunit: Mechanistic insights about its incorporation in lactobacillar SpaCBA pili.

To successfully colonize a host or environment, certain genera and species of Gram-positive bacteria have evolved to utilize the so-called sortase-dependent pilus, a long multi-subunit and non-flagellar surface adhesin. One example of this is Lactobacillus rhamnosus GG, a gut-adapted probiotic strain that produces SpaCBA pili. These structures are covalent hetero-oligomers built from three types of pilin subunit, each with a specific location and function (i.e., backbone SpaA for length, tip SpaC for adhesion, and basal SpaB for anchoring). Functionally, the SpaCBA pilus exhibits a promiscuous affinity for components on intestinal surfaces (e.g., mucus, collagen, and epithelial cells), which is largely attributed to the SpaC subunit. Then again, the basal SpaB pilin, in addition to acting as the terminal subunit during pilus assembly, displays an out of character mucoadhesive function. To address the structural basis of this unusual dual functionality, we reveal the 2.39 â€‹Å resolution crystal structure of SpaB. SpaB consists of one immunoglobulin-like CnaB domain and contains a putative intermolecular isopeptide bond-linking lysine and internal isopeptide bond-asparagine in an FPKN pilin motif within the C-terminal end. Remarkably, we found that a C-terminal stretch of positively charged lysine and arginine residues likely accounts for the atypical mucoadhesiveness of SpaB. Although harboring an autocatalytic triad of residues for a potential internal isopeptide interaction, the SpaB crystal structure lacked the visible electron density for intact bond formation, yet its presence was subsequently confirmed by mass spectral analysis. Finally, we propose a structural model that captures the exclusive basal positioning of SpaB in the SpaCBA pilus.

SpaB, an atypically adhesive basal pilin from the lactobacillar SpaCBA pilus: crystallization and X-ray diffraction analysis.

The SpaB pilin is recognized as the basal subunit of the sortase-dependent SpaCBA pilus, which is known to be produced by the Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensal advocated to have health benefits. Despite seeming to function as an archetypal basal pilin by serving as the terminal subunit in pilus assembly, SpaB also assumes an atypical role as a mucoadhesive protein. To shed light on the structural factors that contribute to this dual functional behaviour, a recombinant form of the L. rhamnosus GG SpaB pilin was produced and purified for crystallization and X-ray diffraction experiments. The crystallization of SpaB remained particularly challenging until the implementation of a three-pronged crystallization approach involving C-terminal tail truncation, surface lysine methylation and magnesium additives. Ultimately, hexagonal crystals of SpaB were produced and were able to diffract to a resolution of 2.4 Å. This crystal form belonged to space group P6522 or P6122, with unit-cell parameters a = b = 51.53, c = 408.22 Å, α = ß = 90.0, γ = 120.0°. Obtaining an interpretable electron-density map via single-wavelength anomalous diffraction (SAD) using iodide-derivative data sets did not succeed owing to the weak anomalous signal. As an alternative, attempts to provide phases by molecular replacement using the iodide-SAD data from SpaB and a collection of distant homology models (<28% sequence identity) are in progress.

Crystal structure of basal pilin SpaE reveals the molecular basis of its incorporation in the lactobacillar SpaFED pilus.

For some Gram-positive genera and species, the long-extended and adhesive sortase-dependent pilus plays an essential role during host colonization, biofilm formation, and immune modulation. Lactobacillus rhamnosus GG is a gut-adapted commensal strain that harbors the operonic genes for the SpaCBA and SpaFED pili, both being comprised of three different protein subunits termed the backbone, tip, and basal pilins. Crystal structures of the backbone pilins (SpaA and SpaD) have recently been solved, and here we describe the high-resolution (1.5 Å) structural determination of the SpaE basal pilin. SpaE consists of two immunoglobulin-like CnaB domains, with each displaying a spontaneously formed internal isopeptide bond, though apparently slow forming in the N-terminal domain. Remarkably, SpaE contains an atypically lengthy unstructured C-terminal tail, along with an YPKN pilin motif peptide, which is normally reserved for backbone subunits. Based on our analysis of the crystal structure data, we provide a molecular model for the basal positioning of the SpaE pilin within the SpaFED pilus.

A SUMOylation-dependent switch of RAB7 governs intracellular life and pathogenesis of Salmonella Typhimurium.

Salmonella Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, S. Typhimurium resides intracellularly in a specialized vesicle, called the Salmonella-containing vacuole (SCV) that utilizes the host endocytic vesicular transport pathway (VTP). Here, we probed the possible role of SUMOylation, a post-translation modification pathway, in SCV biology. Proteome analysis by complex mass-spectrometry (MS/MS) revealed a dramatically altered SUMO-proteome (SUMOylome) in S. Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, in vitro SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7K175R) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by S. Typhimurium ensures availability of long-lived but functionally compromised RAB7, which was beneficial to the pathogen. Overall, this SUMOylation-dependent switch of RAB7 controlled by S. Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond Salmonella-host crosstalk. This article has an associated First Person interview with the first author of the paper.

Bent conformation of a backbone pilin N-terminal domain supports a three-stage pilus assembly mechanism.

Effective colonization of host cells by some Gram-positive bacteria often involves using lengthy, adhesive macromolecular structures called sortase-dependent pili. Among commensals, the gut-adapted Lactobacillus rhamnosus GG strain encodes the operons for two varieties of these pili (SpaCBA and SpaFED), with each structure consisting of backbone, tip, and basal pilin subunits. Although the tertiary structure was recently solved for the backbone subunit (SpaA) of the SpaCBA pilus, no structural information exists for its counterpart in the SpaFED pilus. Here, we report several crystal structures for the SpaD backbone pilin, two of which capture the N-terminal domain in either the closed (linear) or open (bent) conformation. To our knowledge, this is the first observation of the bent conformation in Gram-positive pilin structures. Based on this bent conformation, we suggest a three-stage model, which we call the expose-ligate-seal mechanism, for the docking and assembly of backbone pilins into the sortase-dependent pilus.

Crystallization and X-ray diffraction analysis of SpaE, a basal pilus protein from the gut-adapted Lactobacillus rhamnosus GG.

SpaE is the predicted basal pilin subunit in the sortase-dependent SpaFED pilus from the gut-adapted and commensal Lactobacillus rhamnosus GG. Thus far, structural characterization of the cell-wall-anchoring basal pilins has remained difficult and has been limited to only a few examples from pathogenic genera and species. To gain a further structural understanding of the molecular mechanisms that are involved in the anchoring and assembly of sortase-dependent pili in less harmful bacteria, L. rhamnosus GG SpaE for crystallization was produced by recombinant expression in Escherichia coli. Although several attempts to crystallize the SpaE protein were unsuccessful, trigonal crystals that diffracted to a resolution of 3.1 Šwere eventually produced using PEG 3350 as a precipitant and high protein concentrations. Further optimization with a combination of additives led to the generation of SpaE crystals in an orthorhombic form that diffracted to a higher resolution of 1.5 Å. To expedite structure determination by SAD phasing, selenium-substituted (orthorhombic) SpaE crystals were grown and X-ray diffraction data were collected to 1.8 Šresolution.

New insights about pilus formation in gut-adapted Lactobacillus rhamnosus GG from the crystal structure of the SpaA backbone-pilin subunit.

Thus far, all solved structures of pilin-proteins comprising sortase-assembled pili are from pathogenic genera and species. Here, we present the first crystal structure of a pilin subunit (SpaA) from a non-pathogen host (Lactobacillus rhamnosus GG). SpaA consists of two tandem CnaB-type domains, each with an isopeptide bond and E-box motif. Intriguingly, while the isopeptide bond in the N-terminal domain forms between lysine and asparagine, the one in the C-terminal domain atypically involves aspartate. We also solved crystal structures of mutant proteins where residues implicated in forming isopeptide bonds were replaced. Expectedly, the E-box-substituted E139A mutant lacks an isopeptide bond in the N-terminal domain. However, the C-terminal E269A substitution gave two structures; one of both domains with their isopeptide bonds present, and another of only the N-terminal domain, but with an unformed isopeptide bond and significant conformational changes. This latter crystal structure has never been observed for any other Gram-positive pilin. Notably, the C-terminal isopeptide bond still forms in D295N-substituted SpaA, irrespective of E269 being present or absent. Although E-box mutations affect SpaA proteolytic and thermal stability, a cumulative effect perturbing normal pilus polymerization was unobserved. A model showing the polymerized arrangement of SpaA within the SpaCBA pilus is proposed.

Crystallization and X-ray Crystallographic Analysis of the Adhesive SpaC Pilin Subunit in the SpaCBA Pilus of Gut-adapted Lactobacillus rhamnosus GG.

Gram-positive Lactobacillus rhamnosus GG, a gut-adapted commensalic (and probiotic) strain, is known to express sortase-assembled pili on its cell surface. These SpaCBA-called pili consist of three different types of building blocks; the SpaA backbone-pilin subunit and the SpaB and SpaC ancillary pilins. SpaC is a relatively large (~90kDa) multi-domain fimbrial adhesin, and while it is located primarily at the SpaCBA pilus tip, occasionally, it can also be detected throughout the length of pilus backbone. Functionally, SpaC mainly accounts for SpaCBA pilus-mediated interactions with intestinal mucus, collagen, and human gut epithelial cells. Moreover, SpaC adhesiveness is also perceived to have a causal relationship with SpaCBA pilus-induced host-cell immune responses. In order to improve the mechanistic understanding of SpaC and its adhesive properties by structural investigation, we purified and successfully crystallized a recombinant construct of the near full-length SpaC protein (residues 36-856) in the presence of magnesium ions. X-ray diffraction data were collected to 2.6 Å resolution. The SpaC crystal belongs to the space group P21212 with unit cell parameters a = 116.5, b = 128.3, c = 136.5 Å and contains two molecules in the asymmetric unit. Presence of conserved metal ion-dependent adhesion site containing von Willebrand factor type A domain suggests its likely role in the function of SpaC.

Pilins in gram-positive bacteria: A structural perspective.

Pilins or fimbrilins are a class of proteins found in bacterial surface pilus, a hair-like surface appendage. Both the Gram-negative and -positive bacteria produce pilins to assemble pili on their cell-surface for different purposes including adherence, twitching motility, conjugation, immunomodulation, biofilm formation, and electron transfer. Immunogenic properties of the pilins make them attractive vaccine candidates. The polymerized pilins play a key role in the initiation of host adhesion, which is a critical step for bacterial colonization and infection. Because of their key role in adhesion and exposure on the cell surface, targeting the pilins-mediated adhesion (anti-adhesion therapy) is also seen as a promising alternative approach for preventing and treating bacterial infections, one that may overcome their ever-increasing repertoires of resistance mechanisms. Individual pilins interact with each other non-covalently to assemble the pilus fiber with the help of associated proteins like chaperones and Usher in Gram-negative bacteria. In contrast, the pilins in Gram-positive bacteria often connect with each other covalently, with the help of sortases. Certain unique structural features present on the pilins distinguish them from one another across different bacterial strains, and these dictate their cellular targets and functions. While the structure of pilins has been extensively studied in Gram-negative pathogenic bacteria, the pilins in Gram-positive pathogenic bacteria have been in only during the last decade. Recently, the discovery of pilins in non-pathogenic bacteria, such as Lactobacillus rhamnosus GG, has received great attention, though traditionally the attention was on pathogenic bacteria. This review summarizes and discusses the current structural knowledge of pilins in Gram-positive bacteria with emphasis on those pilins which are sortase substrates.