New Insights into the Biogenesis of Lactobacillus rhamnosus GG Pili and the In Vivo Effects of Pili

The present thesis studied the biogenesis of the pili of Lactobacillus rhamnosus GG, and examined whether pili influence the in vivo persistence and the ability to modulate faecal microbiota in mice and humans. L. rhamnosus GG is one of the most extensively studied lactic acid bacteria, and it is commonly present in so-called probiotic products. Heterotrimeric proteinaceous pili, which are able to bind for example to mucus, collagen and β-lactoglobulin, have been found and characterised on the surface of L. rhamnosus GG during recent years. Pili are formed by the major pilin SpaA, which forms the shaft of the pilus structure, the mucus-binding tip pilin SpaC and basal pilin SpaB. The pili are synthesised on the cell membrane by the pilin-specific transpeptidase sortase C (SrtC), and attached to the cell wall by housekeeping transpeptidase sortase A (SrtA). As the pilus is the major mucus adhesin of L. rhamnosus GG and, according to various studies, a crucial factor in the L. rhamnosus GG adherence and signalling to the host, it was deemed important to characterise the pilus biogenesis pathway and the functions of the produced pili. Random chemical mutagenesis was used to obtain derivatives of L. rhamnosus GG with variable pilus production capacities to study factors affecting pilus biogenesis. Enrichment schemes for the isolation of the derivatives were devised, and consequently 10 pilus-deficient, 13 highly adherent and one pilus-secreting derivative were characterised. Genotypic and phenotypic characterisation of the derivatives revealed that a functional SrtC is essential for pilus biogenesis, as well as the sufficient expression of the major pilin SpaA. Additionally, the role of the insertion sequence-rich genomic region, containing also the pilus gene cluster, in the adaptation of L. rhamnosus GG to varying environments was revealed, as several derivatives were obtained in which the region was deleted. The adhesion capacity of L. rhamnosus GG was found to be increased either due to a mutation in the tip pilin SpaC or as a result of increased pilin production. One of the highly adherent derivatives with high pilin production harboured a mutation in the SpaA pilin, likely increasing the derivative s capacity to secrete this pilus backbone-forming subunit. In addition, a pilus-secreting derivative was characterised, and noted to harbour a mutated housekeeping sortase SrtA, demonstrating its essential role in the attachment of pili to the cell wall. Bioinformatic analysis revealed that the C-terminal sortase recognition motif of pilins, with the sequence LPxTG, was variable between the proteins recognised by pilin-specific transpeptidase SrtC and housekeeping transpeptidase SrtA. This was found not only for the pilins of L. rhamnosus GG, but also for pilins of other sortase-dependent pili-harbouring Gram-positive bacteria, indicating a role for this motif in the regulation of pilus biogenesis. In a subsequent functional characterisation of L. rhamnosus GG pilins, it was demonstrated that SrtC indeed recognised the shaft pilin SpaA through a conserved triple glycine (TG) motif, and was likely to recognise also the tip pilin SpaC, which harbours the same motif. The basal pilin SpaB, containing a single glycine (SG) motif, was recognised by SrtA, leading to the attachment of pilus to the cell wall. Finally, the in vivo effect of the L. rhamnosus GG pili was evaluated in intervention trials in mice and humans. By performing mouse and human intervention trials with one of the described pilus-deficient derivatives, L. rhamnosus GG-PB12 and the parental strain, it was possible to demonstrate that the presence of pili increased the persistence of L. rhamnosus GG in the gastrointestinal tract and led to significant changes in the faecal microbiota composition, especially in humans. Species richness increased significantly due to the consumption of piliated L. rhamnosus GG in the human trial, indicating potential beneficial effects for the host, as a high species richness of gut microbiota has been associated with stability and resilience towards exogenous species. In conclusion, the research described here provides new insights into pilus biogenesis in L. rhamnosus GG and highlights the importance of the pili in the in vivo adherence capacity and gut microbiota modulation ability of L. rhamnosus GG.Tässä väitöskirjassa tutkittiin Lactobacillus rhamnosus GG-bakteerin pilusten, eli tarttumakarvojen, muodostumiseen vaikuttavia tekijöitä. Lisäksi tutkittiin tehostavatko pilukset L. rhamnosus GG:n pysymistä suolistossa sekä sitä, onko piluksilla vaikutusta siihen miten L. rhamnosus GG vaikuttaa suolistomikrobiston koostumukseen sekä ihmisillä että hiirillä. L. rhamnosus GG on yksi laajimmin tutkituista maitohappobakteereista, jolla on niin sanottuja probioottisia ominaisuuksia. L. rhamnosus GG:n pilukset ovat suhteellisen uusi löytö ja niiden karakterisointi on jatkunut viime vuodet. Pilukset koostuvat kolmesta erilaisesta piliini-proteiinista: SpaA:sta, joka muodostaa piluksen varren, SpaC:stä, jota on havaittu pilusten kärjissä ja sivuilla, ja joka on nykytiedon valossa piluksen sitoutumisominaisuuksien kannalta tärkein piliini, sekä SpaB:stä, joka sijoittuu piluksen juureen. Entsyymi nimeltä sortaasi C (SrtC) syntetisoi pilukset solukalvolla, jonka jälkeen sortaasi A (SrtA) kiinnittää pilukset soluseinään. Pilukset pystyvät sitoutumaan mm. mukukseen, kollageeniin ja β-laktoglobuliiniin, ja useiden tutkimusten mukaan pilukset ovat tärkeitä bakteeri-isäntä-vuorovaikutuksen kannalta. Jotta pilusten merkitys voidaan ymmärtää syvällisesti, pilusten muodostumisen, eli biogeneesin, tutkimus on olennaista. Pilusten biogeneesiä voidaan tutkia sellaisten L. rhamnosus GG-kantojen avulla, joiden kyky muodostaa piluksia on muuttunut. Tässä työssä muuntuneita kantoja saatiin eristettyä kemiallisen satunnaismutageneesin ja erityisen rikastusmenetelmän avulla. 10 piluksetonta, 13 tehokkaasti sitoutuvaa ja yksi piluksia erittävä kanta karakterisoitiin sekä geneettisesti että fenotyyppisesti. Piluksettomien kantojen karakterisointi paljasti, että sekä toimiva SrtC-entsyymi että riittävä SpaA-piliinin tuotto, ovat olennaisia pilusten muodostumisen kannalta. Lisäksi havaittiin että genominen alue, jolla pilusten muodostamiseen tarvittavat geenit sijaitsevat, on helposti muuntuva, koska suurella osalla piluksettomista kannoista tämä alue puuttui osittain. Havainto viittaa siihen, että alue on tärkeä L. rhamnosus GG-bakteerin sopeutumisessa vaihteleviin olosuhteisiin. L. rhamnosus GG:n sitoutumiskyvyn lisääntymisen taas havaittiin liittyvän joko mutaatioon SpaC-piliinin geenissä tai lisääntyneeseen piliinien määrään solujen pinnalla. Yhdellä kannoista, joilla piliinien määrä oli lisääntynyt, oli mutaatio SpaA-piliinin geenissä, joka mahdollisesti lisäsi kannan kykyä erittää tätä proteiinia. Tutkimuksessa löydettiin myös piluksia erittävä kanta, jolla SrtA-entsyymin geeni oli mutatoitunut. Löytö vahvisti aiempia havaintoja SrtA:n olennaisesta roolista pilusten kiinnittämisessä soluseinään. Bioinformaattisen analyysin avulla huomattiin että piliinien C-terminaalinen sekvenssi, joka sisältää sortaasi-entsyymien tunnistaman LPxTG-sekvenssin, eroaa riippuen siitä, tunnistaako piliinin piluksia syntetisoiva SrtC vai pilukset soluseinään kiinnittävä SrtA. Sama sekvenssiero huomattiin L. rhamnosus GG:n lisäksi myös muilla Gram-positiivisilla bakteereilla, jotka tuottavat sortaasi-riippuvaisia piluksia. Havainto viittasi siihen, että sekvenssi on tärkeä pilusten biogeneesin säätelyn kannalta. Sekvenssimuotojen funktionaalinen karakterisointi vahvisti, että SrtC tunnistaa piluksen varren muodostavassa SpaA-piliinissä konservoituneen kolmoisglysiini-motiivin (TG), kun taas SrtA tunnistaa SpaB-piliinissä olevan glysiini-motiivin (SG). SpaC-piliini sisältää TG-motiivin samoin kuin SpaA, joten SrtC tunnistaa todennäköisesti myös sen, kuten aiemmin on hypotetisoitu. Tässä työssä tutkittiin pilusten roolia myös L. rhamnosus GG:n pysymisessä suolistossa sekä suolistomikrobiston koostumuksen muokkaamisessa. Asiaa tutkittiin interventiokokein sekä ihmisillä että hiirillä vertailemalla L. rhamnosus GG:n ja piluksettoman kontrollin L. rhamnosus GG-PB12:n vaikutuksia. Kokeissa havaittiin, että pilukset kasvattivat aikaa, jonka L. rhamnosus GG pysyi suolistossa, ja pilusten läsnä ollessa L. rhamnosus GG sai aikaan merkittäviä muutoksia suolistomikrobiston koostumukseen erityisesti ihmisillä. Mikrobiston lajirikkaus kasvoi tilastollisesti merkitsevästi piluksellista L. rhamnosus GG:tä nautittaessa. Tämä viittaa mahdollisiin isäntää hyödyttäviin vaikutuksiin, koska korkea lajirikkaus on yhdistetty mikrobiston tasapainoisuuteen ja vastustuskykyyn ulkoisia haasteita vastaan. Yhteenvetona, esitellyssä väitöstutkimuksessa saadut tulokset lisäävät tietoa L. rhamnosus GG:n pilusten muodostumiseen vaikuttavista tekijöistä sekä korostavat pilusten tärkeyttä L. rhamnosus GG:n in vivo-sitoutumiskyvyssä ja suolistomikrobisto-vaikutuksissa

Genome Sequence of the Butyrate-Producing Anaerobic Bacterium Anaerostipes hadrus PEL 85

Peer reviewe

Effects of Soluble Corn Fiber Alone or in Synbiotic Combination with Lactobacillus rhamnosus GG and the Pilus-Deficient Derivative GG-PB12 on Fecal Microbiota, Metabolism, and Markers of immune Function : A Randomized, Double-Blind, Placebo-Controlled, Crossover Study in Healthy Elderly (Saimes Study)

Background: The aging process leads to a potential decline in immune function and adversely affects the gut microbiota. To date, many in vitro and in vivo studies focused on the application of synbiotics (prebiotics combined with probiotics) as a promising dietary approach to affect gut microbiota composition and improved functioning of the immune system. However, studies using synbiotic preparations often have the limitation that it remains unclear whether any effect observed is a result of the prebiotic or probiotic or a synergistic effect of the combined supplement. Objectives: We investigated the effects of a probiotic Lactobacillus rhamnosus GG and pilus-deficient L. rhamnosus GG-PB12 combined with Promitor (TM) Soluble Corn Fiber (SCF, a candidate prebiotic) on fecal microbiota, metabolism, immunity, and blood lipids in healthy elderly persons. A prospective, double-blind, placebo controlled, randomized, single-centered, crossover study in 40 healthy elderly subjects (aged 60-80 years) was carried out. Volunteers were randomized to consume either probiotic and prebiotic as synbiotic, prebiotic or placebo (maltodextrin) during 3 weeks. Three-week washout periods separated all the treatments. We assessed effects upon blood lipids, glucose, cytokines, natural killer (NK) cell activity, phenotype, and intestinal microbiota composition. SCF decreased IL-6, which was not observed with the synbiotics. Results: Consumption of L. rhamnosus GG combined with SCF increased NK cell activity compared to baseline in females and the older group. In the fecal microbiota analyses, the strongest community shifts were due to L. rhamnosus GG combined with SCF and SCF treatments. L. rhamnosus GG combined with SCF and L. rhamnosus GG-PB12 combined with SCF significantly increased the genus Parabacteroides. L. rhamnosus GG combined with SCF and SCF increased concentrations of Ruminococcaceae Incertae Sedis. Oscillospira and Desulfovibrio slightly decreased in the L. rhamnosus GG combined with SCF group, whereas Desulfovibrio decreased also in the L. rhamnosus GG-PB12 combined with SCF group. L. rhamnosus GG combined with SCF reduced total cholesterol and LDL-cholesterol in volunteers with initially elevated concentrations. C-reactive protein significantly decreased during L. rhamnosus GG-PB12 combined with SCF intervention compared to baseline. Conclusion: In conclusion, the synbiotic combination of L. rhamnosus GG with SCF showed a tendency to promote innate immunity by increasing NK cell activity in elderly women and in 70 to 80-year-old volunteers and decreased TC and LDL-c in hyper-cholesterolemic patients. In addition, L. rhamnosus GG-PB12 combined with SCF demonstrated an increase in NK cell activity compared to SCF alone in older volunteers. We also found significant positive effects on the immune response, evidenced by a decrease of the pro-inflammatory cytokine IL-6. Therefore, dietary intervention with L. rhamnosus GG combined with SCF could be of importance in elderly as an attractive option for enhancement of both the microbial and immune systems.Peer reviewe

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.Peer reviewe

Characterization of Highly Mucus-Adherent Non-GMO Derivatives of Lacticaseibacillus rhamnosus GG

Lacticaseibacillus rhamnosusGG is one of the best studied lactic acid bacteria in the context of probiotic effects.L. rhamnosusGG has been shown to prevent diarrhea in children and adults and has been implicated to have mitigating or preventive effects in several disorders connected to microbiota dysbiosis. The probiotic effects are largely attributed to its adhesive heterotrimeric sortase-dependent pili, encoded by thespaCBA-srtC1gene cluster. Indeed, the strain-specific SpaCBA pili have been shown to contribute to adherence, biofilm formation and host signaling. In this work we set out to generate non-GMO derivatives ofL. rhamnosusGG that adhere stronger to mucus compared to the wild-type strain using chemical mutagenesis. We selected 13 derivatives that showed an increased mucus-adherent phenotype. Deep shotgun resequencing of the strains enabled division of the strains into three classes, two of which revealed SNPs (single nucleotide polymorphisms) in thespaAandspaCgenes encoding the shaft and tip adhesive pilins, respectively. Strikingly, the other class derivatives demonstrated less clear genotype - phenotype relationships, illustrating that pili biogenesis and structure is also affected by other processes. Further characterization of the different classes of derivatives was performed by PacBio SMRT sequencing and RNAseq analysis, which resulted in the identification of molecular candidates driving pilin biosynthesis and functionality. In conclusion, we report on the generation and characterization of three classes of strongly adherentL. rhamnosusGG derivatives that show an increase in adhesion to mucus. These are of special interest as they provide a window on processes and genes driving piliation and its control inL. rhamnosusGG and offer a variety of non-GMO derivatives of this key probiotic strain that are applicable in food products.Peer reviewe

Genotypic and phenotypic diversity of Lactobacillus rhamnosus clinical isolates, their comparison with strain GG and their recognition by complement system

Lactobacillus rhamnosus strains are ubiquitous in fermented foods, and in the human body where they are commensals naturally present in the normal microbiota composition of gut, vagina and skin. However, in some cases, Lactobacillus spp. have been implicated in bacteremia. The aim of the study was to examine the genomic and immunological properties of 16 clinical blood isolates of L. rhamnosus and to compare them to the well- studied L. rhamnosus probiotic strain GG. Blood cultures from bacteremic patients were collected at the Helsinki University Hospital laboratory in 2005-2011 and L. rhamnosus strains were isolated and characterized by genomic sequencing. The capacity of the L. rhamnosus strains to activate serum complement was studied using immunological assays for complement factor C3a and the terminal pathway complement complex (TCC). Binding of complement regulators factor H and C4bp was also determined using radioligand assays. Furthermore, the isolated strains were evaluated for their ability to aggregate platelets and to form biofilms in vitro. Genomic comparison between the clinical L. rhamnosus strains showed them to be clearly different from L. rhamnosus GG and to cluster in two distinct lineages. All L. rhamnosus strains activated complement in serum and none of them bound complement regulators. Four out of 16 clinical blood isolates induced platelet aggregation and/or formed more biofilms than L. rhamnosus GG, which did not display platelet aggregation activity nor showed strong biofilm formation. These findings suggest that clinical L. rhamnosus isolates show considerable heterogeneity but are clearly different from L. rhamnosus GG at the genomic level. All L. rhamnosus strains are still normally recognized by the human complement system.Peer reviewe

Lactobacillus rhamnosus GG Outcompetes Enterococcus faecium via Mucus-Binding Pili : Evidence for a Novel and Heterospecific Probiotic Mechanism

Vancomycin-resistant enterococci (VRE) have become a major nosocomial threat. Enterococcus faecium is of special concern, as it can easily acquire new antibiotic resistances and is an excellent colonizer of the human intestinal tract. Several clinical studies have explored the potential use of beneficial bacteria to weed out opportunistic pathogens. Specifically, the widely studied Lactobacillus rhamnosus strain GG has been applied successfully in the context of VRE infections. Here, we provide new insight into the molecular mechanism underlying the effects of this model probiotic on VRE decolonization. Both clinical VRE isolates and L. rhamnosus GG express pili on their cell walls, which are the key modulators of their highly efficient colonization of the intestinal mucosa. We found that one of the VRE pilus clusters shares considerable sequence similarity with the SpaCBA-SrtC1 pilus cluster of L. rhamnosus GG. Remarkable immunological and functional similarities were discovered between the mucus-binding pili of L. rhamnosus GG and those of the clinical E. faecium strain E1165, which was characterized at the genome level. Moreover, E. faecium strain E1165 bound efficiently to mucus, which may be prevented by the presence of the mucus-binding SpaC protein or antibodies against L. rhamnosus GG or SpaC. These results present experimental support for a novel probiotic mechanism, in which the mucus-binding pili of L. rhamnosus GG prevent the binding of a potential pathogen to the host. Hence, we provide a molecular basis for the further exploitation of L. rhamnosus GG and its pilins for prophylaxis and treatment of VRE infections.Peer reviewe

Characterization of Highly Mucus-Adherent Non-GMO Derivatives of Lacticaseibacillus rhamnosus GG

Lacticaseibacillus rhamnosusGG is one of the best studied lactic acid bacteria in the context of probiotic effects.L. rhamnosusGG has been shown to prevent diarrhea in children and adults and has been implicated to have mitigating or preventive effects in several disorders connected to microbiota dysbiosis. The probiotic effects are largely attributed to its adhesive heterotrimeric sortase-dependent pili, encoded by thespaCBA-srtC1gene cluster. Indeed, the strain-specific SpaCBA pili have been shown to contribute to adherence, biofilm formation and host signaling. In this work we set out to generate non-GMO derivatives ofL. rhamnosusGG that adhere stronger to mucus compared to the wild-type strain using chemical mutagenesis. We selected 13 derivatives that showed an increased mucus-adherent phenotype. Deep shotgun resequencing of the strains enabled division of the strains into three classes, two of which revealed SNPs (single nucleotide polymorphisms) in thespaAandspaCgenes encoding the shaft and tip adhesive pilins, respectively. Strikingly, the other class derivatives demonstrated less clear genotype - phenotype relationships, illustrating that pili biogenesis and structure is also affected by other processes. Further characterization of the different classes of derivatives was performed by PacBio SMRT sequencing and RNAseq analysis, which resulted in the identification of molecular candidates driving pilin biosynthesis and functionality. In conclusion, we report on the generation and characterization of three classes of strongly adherentL. rhamnosusGG derivatives that show an increase in adhesion to mucus. These are of special interest as they provide a window on processes and genes driving piliation and its control inL. rhamnosusGG and offer a variety of non-GMO derivatives of this key probiotic strain that are applicable in food products

Probiotic Gut Microbiota Isolate Interacts with Dendritic Cells via Glycosylated Heterotrimeric Pili

Mapping of the microbial molecules underlying microbiota-host interactions is key to understand how microbiota preserve mucosal homeostasis. A pivotal family of such bacterial molecules are pili. Pili are proteinaceous cell wall appendages with a well-documented role in adhesion, whilst their role in immune interaction with the host is less established. Gram-positive pili are often posttranslationally modified by sortase-specific cleavage reactions and the formation of intramolecular peptide bonds. Here we report glycosylation as a new level of posttranslational modification of sortase-dependent pili of a beneficial microbiota species and its role in immune modulation. We focused on the SpaCBA pili of the model probiotic and beneficial human gut microbiota isolate Lactobacillus rhamnosus GG. A unique combination of molecular techniques, nanoscale mechanical and immunological approaches led to the identification of mannose and fucose residues on the SpaCBA pili. These glycans on the pili are recognized by human dendritic cells via the C-type lectin receptor DC-SIGN, a key carbohydrate-dependent immune tailoring pattern recognition receptor. This specific lectin-sugar interaction is moreover of functional importance and modulated the cytokine response of dendritic cells. This provides insight into the direct role bacterial glycoproteins can play in the immunomodulation of the host. Modification of the complex heterotrimeric pili of a model probiotic and microbiota isolate with mannose and fucose is of importance for the functional interaction with the host immune lectin receptor DC-SIGN on human dendritic cells. Our findings shed light on the yet underappreciated role of glycoconjugates in bacteria-host interactions.Peer reviewe

Selection and characterization of a SpaCBA pilus-secreting food-grade derivative of Lacticaseibacillus rhamnosus GG

Many studies have established the functional properties of Lacticaseibacillus rhamnosus GG, previously known as Lactobacillus rhamnosus GG, marketed worldwide as a probiotic. The extraordinary capacity of L. rhamnosus GG to bind to human mucus and influence the immune system especially stand out. Earlier, we have shown the key role of its SpaCBA sortase-dependent pili encoded by the spaCBA-srtC1 gene cluster herein. These heterotrimeric pili consist of a shaft pilin SpaA, a basal pilin SpaB, and tip pilin SpaC that contains a mucus-binding domain. Here, we set out to characterize a food-grade non-GMO mutant of L. rhamnosus GG, strain PA11, which secretes its pilins, rather than coupling them to the cell surface, due to a defect in the housekeeping sortase A. The sortase-negative strain PA11 was extensively characterized using functional genomics and biochemical approaches and found to secrete the SpaCBA pili into the supernatant. Given the functional importance and uniqueness of the mucus-binding pili of L. rhamnosus GG, strain PA11 offers novel opportunities towards the characterization and further therapeutic application of SpaCBA pili and their low-cost, large-scale production.Peer reviewe