Identification and functional characterisation of a novel surface protein complex of Lactobacillus rhamnosus : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu Campus, New Zealand

Proteins are the most diverse structures on bacterial surfaces; hence they are candidates for species- and strain-specific interactions of bacteria with the host, environment and other microorganisms. In probiotic bacteria, some surface and secreted proteins mediate interactions with the host and may consequently contribute to the health-promoting effects. However, a limited fraction of surface-associated proteins from probiotic bacteria have been functionally characterised to date. A secreted protein of Lactobacillus rhamnosus HN001, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like), was previously shown to bind to HN001 cells, however the nature of its ligand on the surface of the cells was unknown. In this study, a series of binding assays first demonstrated that SpcA binds to a cell wall anchored protein of HN001. Next, the SpcA-“docking” protein, named SpcB, was identified using phage display. SpcB is a 3275-residue cell-surface protein that has all the features of large glycosylated serine-rich adhesins/fibrils from Gram-positive bacteria, including the hallmark glycoprotein signal sequence motif KxYKxGKxW and the cell wall anchor motif LPxTG. The spcA and spcB genes are located in a gene cluster, spcBCDA, which is present in 94 out of 100 strains of L. rhamnosus species and some strains of L. casei and L. paracasei whose genome sequences have been determined, but was absent from other Lactobacillus clades. To confirm the role of SpcB as the SpcA anchor and investigate the roles of these two proteins in surface properties of probiotic L. rhamnosus strains HN001 and GG, stable double-crossover mutations of these two genes were constructed. Binding assays to L. rhamnosus mutant cells confirmed dependence on SpcB in both GG and HN001 strains. Comparison of the wild-type and mutant surface properties suggested that SpcB in GG interferes with biofilm formation and aggregation, while it might contribute to the protective effect against TNFa-mediated disruption of the polarised Caco-2 cell monolayer integrity. Deletion of HN001 spcB or spcA had no effect on functions other than the SpcA binding. Our findings indicate that the roles of a surface protein can vary considerably among the strains of a species, requiring functional data to validate the bioinformatics-based hypotheses

Mucosal adhesion and anti-inflammatory effects of Lactobacillus rhamnosus GG in the human colonic mucosa. A proof-of-concept study

AIM To investigate the adhesion and anti-inflammatory effects of Lactobacillus rhamnosus GG (LGG) in the colonic mucosa of healthy and ulcerative colitis (UC) patients, both in vivo and ex vivo in an organ culture model. METHODS For the ex vivo experiment, a total of 98 patients (68 UC patients and 30 normal subjects) were included. Endoscopic biopsies were collected and incubated with and without LGG or LGG-conditioned media to evaluate the mucosal adhesion and anti-inflammatory effects [reduction of tumor necrosis factor alpha (TNFa) and interleukin (IL)-17 expression] of the bacteria, and extraction of DNA and RNA for quantification by real-time (RT)-PCR occurred after the incubation. A dose-response study was performed by incubating biopsies at "regular", double and 5 times higher doses of LGG. For the in vivo experiment, a total of 42 patients (20 UC patients and 22 normal controls) were included. Biopsies were taken from the colons of normal subjects who consumed a commercial formulation of LGG for 7 d prior to the colonoscopy, and the adhesion of the bacteria to the colonic mucosa was evaluated by RT-PCR and compared with that of control biopsies from patients who did not consume the formulation. LGG adhesion and TNFa and IL-17 expression were compared between UC patients who consumed a regular or double dose of LGG supplementation prior to colonoscopy. RESULTS In the ex vivo experiment, LGG showed consistent adhesion to the distal and proximal colon in normal subjects and UC patients, with a trend towards higher concentrations in the distal colon, and in UC patients, adhesion was similar in biopsies with active and quiescent inflammation. In addition, bioptic samples from UC patients incubated with LGG conditioned media (CM) showed reduced expression of TNFa and IL-17 compared with the corresponding expression in controls (P < 0.05). Incubation with a double dose of LGG increased mucosal adhesion and the anti-inflammatory effects (P < 0.05). In the in vivo experiment, LGG was detectable only in the colon of patients who consumed the LGG formulation, and bowel cleansing did not affect LGG adhesion. UC patients who consumed the double LGG dose had increased mucosal concentrations of the bacteria and reduced TNFa and IL-17 expression compared with patients who consumed the regular dose (48% and 40% reduction, respectively, P < 0.05). CONCLUSION In an ex vivo organ culture model, LGG showed consistent adhesion and anti-inflammatory effects. Colonization by LGG after consumption for a week was demonstrated in vivo in the human colon. Increasing the administered dose increased the adhesion and effectiveness of the bacteria. For the first time, we demonstrated that LGG effectively adheres to the colonic mucosa and exerts antiinflammatory effects, both ex vivo and in vivo

Effect of Lactobacillus rhamnosus HN001 and Bifidobacterium longum BB536 on the healthy gut microbiota composition at phyla and species level: a preliminary study

AIM: To evaluate the ability of Lactobacillus rhamnosus HN001 and Bifidobacterium longum BB536 to colonize the intestinal environment of healthy subjects and modify the gut microbiota composition. METHODS: Twenty healthy Italian volunteers, eight males and twelve females, participated in the study. Ten subjects took a sachet containing 4 × 109 colony-forming units (CFU) of Bifidobacterium longum BB536 and 109 CFU of Lactobacillus rhamnosus HN001, 30 min before breakfast (pre-prandial administration), while ten subjects took a sachet of probiotic product 30 min after breakfast (post-prandial administration). The ability of Lactobacillus rhamnosus HN001 and Bifidobacterium longum BB536 to colonize human gut microbiota was assessed by means of quantitative real-time PCR, while changes in gut microbiota composition were detected by using Ion Torrent Personal Genome Machine. RESULTS: Immediately after 1-mo of probiotic administration, B. longum BB536 and L. rhamnosus HN001 load was increased in the majority of subjects in both pre-prandial and post-prandial groups. This increase was found also 1 mo after the end of probiotic oral intake in both groups, if compared to samples collected before probiotic consumption. At phyla level a significant decrease in Firmicutes abundance was detected immediately after 1-mo of B. longum BB536 and L. rhamnosus HN001 oral intake. This reduction persisted up to 1 mo after the end of probiotic oral intake together with a significant decrease of Proteobacteria abundance if compared to samples collected before probiotic administration. Whereas, at species level, a higher abundance of Blautia producta, Blautia wexlerae and Haemophilus ducrey was observed, together with a reduction of Holdemania filiformis, Escherichia vulneris, Gemmiger formicilis and Streptococcus sinensis abundance. In addition, during follow-up period we observed a further reduction in Escherichia vulneris and Gemmiger formicilis, together with a decrease in Roseburia faecis and Ruminococcus gnavus abundance. Conversely, the abundance of Akkermansia muciniphila was increased if compared to samples collected at the beginning of the experimental time course. CONCLUSION: B. longum BB536 and L. rhamnosus HN001 showed the ability to modulate the gut microbiota composition, leading to a significant reduction of potentially harmful bacteria and an increase of beneficial ones. Further studies are needed to better understand the specific mechanisms involved in gut microbiota modulation

Antifungal defense of probiotic Lactobacillus rhamnosus GG is mediated by blocking adhesion and nutrient depletion

Data Availability: All relevant data are available from the Gene Expression Omnibus at the following accession number: GSE97755. Funding: This work was funded by the German Research Council (DFG) Graduation College 685, Dr. Jekyll and Mr. Hyde: A systems approach to the therapy of nosocomial infections caused by Candida albicans: a commensal organism switches to a deadly pathogen/ PTJ (FKZ: 0315409BBMBF), the Dr. Manfred Plempel-foundation, the Dr. Siegried Stettendorf-Foundation, the InfectERA Program (FunComPath; BMBF FKZ 031L0001A), the Integrated Research and Treatment Center for Sepsis Control and Care (CSCC) project CanBac (BMBF, FKZ: 01EO1002), and the German Research Council (DFG) GZ:HE7565/1-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Tolerance and safety of the potentially probiotic strain Lactobacillus rhamnosus PRSF-L477 : a randomised, double-blind placebo-controlled trial in healthy volunteers

In Europe, the species Lactobacillus rhamnosus is currently on the Qualified Presumption of Safety list used by the European Food Safety Authority (EFSA) for internal safety assessment, but according to the EFSA the species should remain a topic of surveillance. In the present study, the safety and tolerance of the potentially probiotic strain L. rhamnosus PRSF-L477 was investigated in a placebo-controlled double-blind volunteer trial following FAO/WHO guidelines. A total of thirty-four subjects received daily doses of 1 x 10(11) colony-forming units (cfu) of L. rhamnosus PRSF-L477 (n 17) or placebo (n 17) for a period of 3 weeks, followed by a wash-out period of another 3 weeks. A questionnaire on gastrointestinal tolerance and a diary was kept daily to record compliance throughout these 6 weeks. Faecal and blood samples were collected for microbiological and haematological analysis. The recorded gastrointestinal symptoms, defecation frequency and stool consistency were not influenced indicating that L. rhamnosus PRSF-L477 was well tolerated. The species L. rhamnosus was detected in the faeces of sixteen out of seventeen subjects of the probiotic group during the intervention period. Using pulsed-field gel electrophoresis, re-isolates of L. rhamnosus PRSF-L477 were confirmed in nine of these subjects. Antibiotic susceptibility profiles of these re-isolates were unchanged compared with PRSF-L477. No clinically relevant changes in blood parameters such as liver and kidney function and no serious adverse events appeared during and after administration. Therefore, we conclude that L. rhamnosus PRSF-L477 can safely be administrated to healthy subjects at a daily dose of 1 x 10(11) cfu

Long-term Lactobacillus rhamnosus BMX 54 application to restore a balanced vaginal ecosystem: a promising solution against HPV-infection

Background: Over recent years, a growing interest has developed in microbiota and in the concept of maintaining a special balance between Lactobacillus and other bacteria species in order to promote women's well-being. The aim of our study was to confirm that vaginal Lactobacilli long-lasting implementation in women with HPV-infections and concomitant bacterial vaginosis or vaginitis might be able to help in solving the viral infection, by re-establishing the original eubiosis.Methods: A total of 117 women affected by bacterial vaginosis or vaginitis with concomitant HPV-infections were enrolled at Department of Gynecological Obstetrics and Urological Sciences, La Sapienza University, Rome, Italy between February 2015 and March 2016. Women were randomized in two groups, standard treatment (metronidazole 500 mg twice a day for 7 days or fluconazole 150 mg orally once a day for 2 consecutive days) plus short-term (3 months) vaginal Lactobacillus implementation (group 1, short probiotics treatment protocol group, n = 60) versus the same standard treatment plus long-lasting (6 months) vaginal Lactobacillus rhamnosus BMX 54 administration (group 2, treatment group, n = 57).Results: After a median follow up of 14 months (range 9-30 months) the chance to solve HPV-related cytological anomalies was twice higher in probiotic long-term users (group 2) versus short probiotics implementation group (group 1) (79.4% vs 37.5%, p = 0.041). Moreover, a total HPV-clearance was shown in 11.6% of short schedule probiotics implementation patients compared to a percentage of 31.2% in vaginal Lactobacilli long term users (p = 0.044), assessed as negative HPV-DNA test documented at the end of the study period.Conclusions: The consistent percentage of clearance of PAP-smear abnormalities and HPV-clearance obtained in long-term treatment group has been interestingly high and encouraging. Obviously, larger and randomized studies are warranted to confirm these encouraging results, but we believe that eubiosis re-establishment is the key to tackle effectively even HPV-infection

Are the immunomodulatory properties of Lactobacillus rhamnosus CRL1505 peptidoglycan common for all Lactobacilli during respiratory infection in malnourished mice?

Previously, we reported that Lactobacillus rhamnosus CRL1505 peptidoglycan (PG05) improves the innate immune response in immunocompromised-malnourished mice after Streptococcus pneumoniae infection. This study extends those previous findings by demonstrating that the dietary recovery of malnourished mice with nasal administration of PG05 improves not only the innate immune response but the respiratory and systemic adaptive humoral response as well. PG05 enhanced the Th2 response, the recovery of B cells, and the concentration and opsonophagocytic activity of anti-pneumococcal antibodies. In addition, by performing comparative studies with the peptidoglycans from lactobacilli of the same species (L. rhamnosus CRL534) or with similar immunomodulatory properties (L. plantarum CRL1506), we demonstrated here that PG05 has unique immunomodulatory properties that cannot be extended to peptidoglycans from other probiotic strains. However, the knowledge of the molecular characteristics of PG05 is indispensable to understand immunomodulatory abilities of L. rhamnosus CRL1505.Fil: Kolling, Yanina Noralí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Salva, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Villena, Julio Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Alvarez, Gladis Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

Incorporating a mucosal environment in a dynamic gut model results in a more representative colonization by lactobacilli

To avoid detrimental interactions with intestinal microbes, the human epithelium is covered with a protective mucus layer that traps host defence molecules. Microbial properties such as adhesion to mucus further result in a unique mucosal microbiota with a great potential to interact with the host. As mucosal microbes are difficult to study in vivo, we incorporated mucin-covered microcosms in a dynamic in vitro gut model, the simulator of the human intestinal microbial ecosystem (SHIME). We assessed the importance of the mucosal environment in this M-SHIME (mucosal-SHIME) for the colonization of lactobacilli, a group for which the mucus binding domain was recently discovered. Whereas the two dominant resident Lactobacilli, Lactobacillus mucosae and Pediococcus acidilactici, were both present in the lumen, L. mucosae was strongly enriched in mucus. As a possible explanation, the gene encoding a mucus binding (mub) protein was detected by PCR in L. mucosae. Also the strongly adherent Lactobacillus rhamnosus GG (LGG) specifically colonized mucus upon inoculation. Short-term assays confirmed the strong mucin-binding of both L. mucosae and LGG compared with P. acidilactici. The mucosal environment also increased long-term colonization of L. mucosae and enhanced its stability upon antibiotic treatment (tetracycline, amoxicillin and ciprofloxacin). Incorporating a mucosal environment thus allowed colonization of specific microbes such as L. mucosae and LGG, in correspondence with the in vivo situation. This may lead to more in vivo-like microbial communities in such dynamic, long-term in vitro simulations and allow the study of the unique mucosal microbiota in health and disease

Dietary Prebiotics and Bioactive Milk Fractions Improve NREM Sleep, Enhance REM Sleep Rebound and Attenuate the Stress-Induced Decrease in Diurnal Temperature and Gut Microbial Alpha Diversity.

Severe, repeated or chronic stress produces negative health outcomes including disruptions of the sleep/wake cycle and gut microbial dysbiosis. Diets rich in prebiotics and glycoproteins impact the gut microbiota and may increase gut microbial species that reduce the impact of stress. This experiment tested the hypothesis that consumption of dietary prebiotics, lactoferrin (Lf) and milk fat globule membrane (MFGM) will reduce the negative physiological impacts of stress. Male F344 rats, postnatal day (PND) 24, received a diet with prebiotics, Lf and MFGM (test) or a calorically matched control diet. Fecal samples were collected on PND 35/70/91 for 16S rRNA sequencing to examine microbial composition and, in a subset of rats; Lactobacillus rhamnosus was measured using selective culture. On PND 59, biotelemetry devices were implanted to record sleep/wake electroencephalographic (EEG). Rats were exposed to an acute stressor (100, 1.5 mA, tail shocks) on PND 87 and recordings continued until PND 94. Test diet, compared to control diet, increased fecal Lactobacillus rhamnosus colony forming units (CFU), facilitated non-rapid eye movement (NREM) sleep consolidation (PND 71/72) and enhanced rapid eye movement (REM) sleep rebound after stressor exposure (PND 87). Rats fed control diet had stress-induced reductions in alpha diversity and diurnal amplitude of temperature, which were attenuated by the test diet (PND 91). Stepwise multiple regression analysis revealed a significant linear relationship between early-life Deferribacteres (PND 35) and longer NREM sleep episodes (PND 71/72). A diet containing prebiotics, Lf and MFGM enhanced sleep quality, which was related to changes in gut bacteria and modulated the impact of stress on sleep, diurnal rhythms and the gut microbiota