Piliation of Lactobacillus rhamnosus GG Promotes Adhesion,Phagocytosis, and Cytokine Modulation in Macrophages

Recently, spaCBA-encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.     

The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG

Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoid-shaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.     

Pilus Biogenesis in Lactococcus lactis: Molecular Characterization and Role in Aggregation and Biofilm Formation

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.     

The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin

Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0–200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis.     

Processed VirB2 Is the Major Subunit of the Promiscuous Pilus of Agrobacterium tumefaciens

Previous studies have implicated the obligatory requirement for the vir regulon (or “virulon”) of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced A. tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19°C as compared with 28°C, an observation that parallels the effect of low temperature on the production of vir gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107–1109, 1996). Export of the processed VirB2 requires other virB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the “T pilus” of A. tumefaciens.     

Evidence for the Sialylation of PilA,the PI-2a Pilus-Associated Adhesin of Streptococcus agalactiae Strain NEM316

Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.     

Characterization of a 20-kDa pilus protein expressed by a diarrheogenic strain of non-O1/non-O139 Vibrio cholerae

A diarrheogenic strain of non-O1/non-O139 Vibrio cholerae (10325) belonging to serogroup O34 was earlier shown to express a new type of pilus composed of a 20-kDa subunit protein. Amino-terminal sequence data (determined up to 20 amino acid residues) of this protein showed it to be different from the subunit proteins of other known types of pili of V. cholerae. On the other hand, it showed complete homology with the corresponding sequence of a 22-kDa outer membrane protein (OmpW) of V. cholerae. Expression of 10325 pili was favored in AKI rather than in NB medium and at 30°C rather than at 37°C. Further, cultural conditions favoring pilus expression also enhanced autoagglutination and adherence properties of strain 10325. An antiserum to the 20-kDa protein induced passive protection against challenge with the parent organism 10325, but not against V. cholerae O1 strains. Such protection was shown to be mediated by inhibition of intestinal colonization in vivo.     

Carbohydrate catabolic flexibility in the mammalian intestinal commensal <Emphasis Type="Italic">Lactobacillus ruminis</Emphasis> revealed by fermentation studies aligned to genome annotations

Background

Lactobacillus ruminis is a poorly characterized member of the Lactobacillus salivarius clade that is part of the intestinal microbiota of pigs, humans and other mammals. Its variable abundance in human and animals may be linked to historical changes over time and geographical differences in dietary intake of complex carbohydrates.

Results

In this study, we investigated the ability of nine L. ruminis strains of human and bovine origin to utilize fifty carbohydrates including simple sugars, oligosaccharides, and prebiotic polysaccharides. The growth patterns were compared with metabolic pathways predicted by annotation of a high quality draft genome sequence of ATCC 25644 (human isolate) and the complete genome of ATCC 27782 (bovine isolate). All of the strains tested utilized prebiotics including fructooligosaccharides (FOS), soybean-oligosaccharides (SOS) and 1,3:1,4-β-D-gluco-oligosaccharides to varying degrees. Six strains isolated from humans utilized FOS-enriched inulin, as well as FOS. In contrast, three strains isolated from cows grew poorly in FOS-supplemented medium. In general, carbohydrate utilisation patterns were strain-dependent and also varied depending on the degree of polymerisation or complexity of structure. Six putative operons were identified in the genome of the human isolate ATCC 25644 for the transport and utilisation of the prebiotics FOS, galacto-oligosaccharides (GOS), SOS, and 1,3:1,4-β-D-Gluco-oligosaccharides. One of these comprised a novel FOS utilisation operon with predicted capacity to degrade chicory-derived FOS. However, only three of these operons were identified in the ATCC 27782 genome that might account for the utilisation of only SOS and 1,3:1,4-β-D-Gluco-oligosaccharides.

Conclusions

This study has provided definitive genome-based evidence to support the fermentation patterns of nine strains of Lactobacillus ruminis, and has linked it to gene distribution patterns in strains from different sources. Furthermore, the study has identified prebiotic carbohydrates with the potential to promote L. ruminis growth in vivo.

     

Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis

In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.     

Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG is a human intestinal isolate that has been studied intensively because of its probiotic properties. We have previously shown that L. rhamnosus GG produces proteinaceous pili that earlier had been observed only in Gram-positive pathogens (M. Kankainen et al., Proc. Natl. Acad. Sci. U. S. A. 106:17193-17198, 2009). These pili were found to be encoded by the spaCBA gene cluster, and the pilus-associated SpaC pilin was shown to confer on the cells a mucus-binding ability. In addition to the spaCBA cluster, another putative pilus cluster, spaFED, was predicted from the L. rhamnosus GG genome sequence. Herein, we show that only SpaCBA pili are produced by L. rhamnosus, and we describe a detailed analysis of cell wall-associated and affinity-purified SpaCBA pili by Western blotting and immunogold electron microscopy. Our results indicate that SpaCBA pili are heterotrimeric protrusions with a SpaA subunit as the shaft-forming major pilin. Only a few SpaB subunits could be observed in pilus fibers. Instead, SpaB pilins were found at pilus bases, as assessed by immunogold double labeling of thin sections of cells, suggesting that SpaB is involved in the termination of pilus assembly. The SpaC adhesin was present along the whole pilus length at numbers nearly equaling those of SpaA. The relative amount and uniform distribution of SpaC within pili not only makes it possible to exert both long-distance and intimate contact with host tissue but also provides mucus-binding strength, which explains the prolonged intestinal residency times observed for L. rhamnosus GG compared to that of nonpiliated lactobacilli.     

Crystal Structure of the Minor Pilin CofB,the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli

Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 Å crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system.     

Biogenesis and adhesion of type 1 and P pili

BackgroundUropathogenic Escherichia coli (UPEC) cause urinary tract infections (UTIs) in approximately 50% of women. These bacteria use type 1 and P pili for host recognition and attachment. These pili are assembled by the chaperone-usher pathway of pilus biogenesis.Scope of reviewThe review examines the biogenesis and adhesion of the UPEC type 1 and P pili. Particular emphasis is drawn to the role of the outer membrane usher protein. The structural properties of the complete pilus are also examined to highlight the strength and functionality of the final assembly.Major conclusionsThe usher orchestrates the sequential addition of pilus subunits in a defined order. This process follows a subunit-incorporation cycle which consists of four steps: recruitment at the usher N-terminal domain, donor-strand exchange with the previously assembled subunit, transfer to the usher C-terminal domains and translocation of the nascent pilus.Adhesion by the type 1 and P pili is strengthened by the quaternary structure of their rod sections. The rod is endowed with spring-like properties which provide mechanical resistance against urine flow. The distal adhesins operate differently from one another, targeting receptors in a specific manner.The biogenesis and adhesion of type 1 and P pili are being therapeutically targeted, and efforts to prevent pilus growth or adherence are described.General significanceThe combination of structural and biochemical study has led to the detailed mechanistic understanding of this membrane spanning nano-machine. This can now be exploited to design novel drugs able to inhibit virulence. This is vital in the present era of resurgent antibiotic resistance. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.     

The three-dimensional structure of CFA/I adhesion pili: traveler's diarrhea bacteria hang on by a spring

To survive the harsh environment of a churning intestinal tract, bacteria attach to the host epithelium via thin fibers called pili (or fimbriae). Enterotoxigenic Escherichia coli bacteria expressing colonization factor antigen I (CFA/I) pili and related pili are the most common known bacterial cause of diarrheal disease, including traveler's diarrhea. CFA/I pili, assembled via the alternate chaperone pathway, are essential for binding and colonization of the small bowel by these pathogenic bacteria. Herein, we elucidate unique structural features of CFA/I pili that appear to optimize their function as bacterial tethers in the intestinal tract. Using transmission electron microscopy of negatively stained samples in combination with iterative three-dimensional helical reconstruction methods for image processing, we determined the structure of the CFA/I pilus filament. Our results indicate that strong end-to-end protein interactions and weak interactions between the coils of a sturdy spring-like helix provide the combination of strength, stability, and flexibility required to sustain bacterial adhesion and incite intestinal disease. We propose that CFA/I pili behave like a spring to maintain attachment to the gut lining during vortex mixing and downward flow of the intestinal contents, thereby persisting long enough for these bacteria to colonize the host epithelium and cause enteric disease.     

Contact Stimulation of Tgl and Type IV Pili in Myxococcus xanthus

Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl⁺ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.     

C-di-GMP Regulates Motile to Sessile Transition by Modulating MshA Pili Biogenesis and Near-Surface Motility Behavior in Vibrio cholerae

In many bacteria, including Vibrio cholerae, cyclic dimeric guanosine monophosphate (c-di-GMP) controls the motile to biofilm life style switch. Yet, little is known about how this occurs. In this study, we report that changes in c-di-GMP concentration impact the biosynthesis of the MshA pili, resulting in altered motility and biofilm phenotypes in V. cholerae. Previously, we reported that cdgJ encodes a c-di-GMP phosphodiesterase and a ΔcdgJ mutant has reduced motility and enhanced biofilm formation. Here we show that loss of the genes required for the mannose-sensitive hemagglutinin (MshA) pilus biogenesis restores motility in the ΔcdgJ mutant. Mutations of the predicted ATPase proteins mshE or pilT, responsible for polymerizing and depolymerizing MshA pili, impair near surface motility behavior and initial surface attachment dynamics. A ΔcdgJ mutant has enhanced surface attachment, while the ΔcdgJmshA mutant phenocopies the high motility and low attachment phenotypes observed in a ΔmshA strain. Elevated concentrations of c-di-GMP enhance surface MshA pilus production. MshE, but not PilT binds c-di-GMP directly, establishing a mechanism for c-di-GMP signaling input in MshA pilus production. Collectively, our results suggest that the dynamic nature of the MshA pilus established by the assembly and disassembly of pilin subunits is essential for transition from the motile to sessile lifestyle and that c-di-GMP affects MshA pilus assembly and function through direct interactions with the MshE ATPase.     

Exopolysaccharide Produced by Lactobacillus Plantarum Induces Maturation of Dendritic Cells in BALB/c Mice

Lactobacillus plantarum (L. plantarum) exopolysaccharide (EPS) is an important bioactive component in fermented functional foods. However, there is a lack of data concerning the effects of L. plantarum EPS on maturation of mouse dendritic cells (DCs). In this study, we purified L. plantarum EPS and examined its effects on cytokines production by dendritic cells in serum and intestinal fluid of BALB/c mice, then investigated its effects on phenotypic and functional maturation of mouse bone marrow-derived dendritic cells (BMDCs). Cytokines (nitric oxide, IL-12p70, IL-10 and RANTES) in serum and intestinal fluid were analyzed by enzyme linked immunosorbent assay (ELISA) after the mice received EPS for 2, 5 and 7 days, respectively. DCs derived from bone marrow of BALB/c mouse were treated with EPS, then the phenotypic maturation of BMDCs was analyzed using flow cytometer and the functional maturation of BMDCs was analyzed by ELISA, and, lastly, mixed lymphocyte proliferation was performed. We found the molecular weight of purified EPS was approximately 2.4×10⁶ Da and it was composed of ribose, rhamnose, arabinose, xylose, mannose, glucose and galactose in a molar ratio of 2:1:1:10:4:205:215. We observed that L. plantarum EPS enriched production of nitric oxide, IL-12p70 and RANTES, and decreased the secretion of IL-10 in the serum or intestinal fluid as well as in the supernatant of DCs treated with the EPS. The EPS also up-regulated the expression of MHC II and CD86 on DCs surface and promoted T cells to proliferate in vitro. Our data provide direct evidence to suggest that L. plantarum EPS can effectively induce maturation of DCs in mice.     

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.     

Structure and function of the adhesive type IV pilus of Sulfolobus acidocaldarius

Archaea display a variety of type IV pili on their surface and employ them in different physiological functions. In the crenarchaeon Sulfolobus acidocaldarius the most abundant surface structure is the aap pilus (a rchaeal a dhesive p ilus). The construction of in frame deletions of the aap genes revealed that all the five genes (aapA, aapX, aapE, aapF, aapB) are indispensible for assembly of the pilus and an impact on surface motility and biofilm formation was observed. Our analyses revealed that there exists a regulatory cross‐talk between the expression of aap genes and archaella (formerly archaeal flagella) genes during different growth phases. The structure of the aap pilus is entirely different from the known bacterial type IV pili as well as other archaeal type IV pili. An aap pilus displayed 3 stranded helices where there is a rotation per subunit of ～ 138° and a rise per subunit of ～ 5.7 Å. The filaments have a diameter of ～ 110 Å and the resolution was judged to be ～ 9 Å. We concluded that small changes in sequence might be amplified by large changes in higher‐order packing. Our finding of an extraordinary stability of aap pili possibly represents an adaptation to harsh environments that S. acidocaldarius encounters.