Oral and non-oral lichen planus show genetic heterogeneity and differential risk for autoimmune disease and oral cancer.

Lichen planus (LP) is a T-cell-mediated inflammatory disease affecting squamous epithelia in many parts of the body, most often the skin and oral mucosa. Cutaneous LP is usually transient and oral LP (OLP) is most often chronic, so we performed a large-scale genetic and epidemiological study of LP to address whether the oral and non-oral subgroups have shared or distinct underlying pathologies and their overlap with autoimmune disease. Using lifelong records covering diagnoses, procedures, and clinic identity from 473,580 individuals in the FinnGen study, genome-wide association analyses were conducted on carefully constructed subcategories of OLP (n = 3,323) and non-oral LP (n = 4,356) and on the combined group. We identified 15 genome-wide significant associations in FinnGen and an additional 12 when meta-analyzed with UKBB (27 independent associations at 25 distinct genomic locations), most of which are shared between oral and non-oral LP. Many associations coincide with known autoimmune disease loci, consistent with the epidemiologic enrichment of LP with hypothyroidism and other autoimmune diseases. Notably, a third of the FinnGen associations demonstrate significant differences between OLP and non-OLP. We also observed a 13.6-fold risk for tongue cancer and an elevated risk for other oral cancers in OLP, in agreement with earlier reports that connect LP with higher cancer incidence. In addition to a large-scale dissection of LP genetics and comorbidities, our study demonstrates the use of comprehensive, multidimensional health registry data to address outstanding clinical questions and reveal underlying biological mechanisms in common but understudied diseases.

A machine-learning method for biobank-scale genetic prediction of blood group antigens.

A key element for successful blood transfusion is compatibility of the patient and donor red blood cell (RBC) antigens. Precise antigen matching reduces the risk for immunization and other adverse transfusion outcomes. RBC antigens are encoded by specific genes, which allows developing computational methods for determining antigens from genomic data. We describe here a classification method for determining RBC antigens from genotyping array data. Random forest models for 39 RBC antigens in 14 blood group systems and for human platelet antigen (HPA)-1 were trained and tested using genotype and RBC antigen and HPA-1 typing data available for 1,192 blood donors in the Finnish Blood Service Biobank. The algorithm and models were further evaluated using a validation cohort of 111,667 Danish blood donors. In the Finnish test data set, the median (interquartile range [IQR]) balanced accuracy for 39 models was 99.9 (98.9-100)%. We were able to replicate 34 out of 39 Finnish models in the Danish cohort and the median (IQR) balanced accuracy for classifications was 97.1 (90.1-99.4)%. When applying models trained with the Danish cohort, the median (IQR) balanced accuracy for the 40 Danish models in the Danish test data set was 99.3 (95.1-99.8)%. The RBC antigen and HPA-1 prediction models demonstrated high overall accuracies suitable for probabilistic determination of blood groups and HPA-1 at biobank-scale. Furthermore, population-specific training cohort increased the accuracies of the models. This stand-alone and freely available method is applicable for research and screening for antigen-negative blood donors.

Sperm Physiological Response to Female Serum-Potential New Insights into the Reproductive Incompatibility Diagnostics.

Infertility is assumed to arise exclusively from male- and female-dependent pathological factors. However, recent studies have indicated that reproductive failure may also result from the reproductive incompatibility of the partners. Selection against such incompatibilities likely occurs via female-derived reproductive secretions, including follicular fluid (FF), that mediate gamete-level mate choice towards the sperm of specific males. To facilitate potential development of diagnostic tests for human reproductive incompatibility, we examined whether sperm physiological response to female serum indicate male-female compatibility in the presence of FF. We performed a full-factorial experiment, in which the sperm of 10 males were treated with the FF and serum of 6 healthy females. We found that sperm motility and viability in both biofluids were highly similar and that in 70% of the males, sperm serum treatment predicted male-female compatibility. We also identified male human leucocyte antigen (HLA) alleles and female (FF and serum) anti-HLA antibodies and tested whether the number of allele-antibody matches predict sperm physiological response to female fluids. However, no association was found between measured sperm traits and the number of allele-antibody matches. Overall, the present results may open novel possibilities for the future development of reproductive incompatibility tests and may pave the way towards more accurate infertility diagnostics and treatments.

Genomic convergence between Akkermansia muciniphila in different mammalian hosts.

BACKGROUND: Akkermansia muciniphila is a member of the human gut microbiota where it resides in the mucus layer and uses mucin as the sole carbon, nitrogen and energy source. A. muciniphila is the only representative of the Verrucomicrobia phylum in the human gut. However, A. muciniphila 16S rRNA gene sequences have also been found in the intestines of many vertebrates. RESULTS: We detected A. muciniphila-like bacteria in the intestines of animals belonging to 15 out of 16 mammalian orders. In addition, other species belonging to the Verrucomicrobia phylum were detected in fecal samples. We isolated 10 new A. muciniphila strains from the feces of chimpanzee, siamang, mouse, pig, reindeer, horse and elephant. The physiology and genome of these strains were highly similar in comparison to the type strain A. muciniphila MucT. Overall, the genomes of the new strains showed high average nucleotide identity (93.9 to 99.7%). In these genomes, we detected considerable conservation of at least 75 of the 78 mucin degradation genes that were previously detected in the genome of the type strain MucT. CONCLUSIONS: The low genomic divergence observed in the new strains may indicate that A. muciniphila favors mucosal colonization independent of the differences in hosts. In addition, the conserved mucus degradation capability points towards a similar beneficial role of the new strains in regulating host metabolic health.

Structural dissimilarity of partners' immune genes increases sperm viability in women's reproductive tract.

Human leucocyte antigen (HLA) genes appear to mediate pre- and post-copulatory mate choice towards HLA-dissimilar ('compatible') partners. However, since genetically distinct alleles often have similar immunogenic properties, genetic dissimilarity is not necessarily an accurate predictor of the functional compatibility of HLA alleles and, hence, may not reflect partners' true compatibility. Furthermore, it has remained unclear whether other genes of the immune system could also play a role in male-female compatibility. We studied whether the immunoglobulin binding regions (eplets) of HLA molecules and the immunoglobulin structural dissimilarity of the partners affect their gamete-level compatibility. We exposed sperm of multiple men to follicular fluid or cervical mucus of multiple women and tested whether sperm viability in these reproductive secretions was influenced by HLA eplet and immunoglobulin structural dissimilarity between partners. We found that eplet dissimilarity positively affects sperm viability in follicular fluid, whereas immunoglobulin dissimilarity enhanced sperm viability in cervical mucus. Together, these findings indicate that structural characteristics of both HLA alleles and immunoglobulins may facilitate cryptic female choice towards immunologically compatible partners. Our results, thus, indicate that partners' genetic compatibility may have wider immunological basis than traditionally has been assumed. Relative contribution of different immunogenetic factors to overall compatibility of the reproductive partners needs to be clarified in future studies.

HLA-DQ and HLA-DRB1 alleles associated with Henoch-Schönlein purpura nephritis in Finnish pediatric population: a genome-wide association study.

BACKGROUND: The pathophysiology of Henoch-Schönlein purpura (HSP) is still unclear, but several findings suggest that genetic factors may influence disease susceptibility. We aimed to perform a genome-wide association study (GWAS) in pediatric HSP patients with an emphasis on severe HSP nephritis. METHODS: The study included 46 HSP patients, 42 of whom had undergone kidney biopsy. Forty-nine pediatric patients with an inflammatory bowel disease (IBD) served as an autoimmune disease control group while Finnish bone marrow and blood donors represented the general reference population (n = 18,757). GWAS was performed for HSP and IBD samples in a case-control manner against the reference population. The analysis also included imputation of human leukocyte antigen (HLA) alleles. RESULTS: GWAS analysis in HSP revealed several polymorphisms from the HLA region that surpassed the genome-wide significance level. Three HLA class II alleles were also significantly more frequent in HSP than in the reference population: DQA1*01:01, DQB1*05:01, and DRB1*01:01. Haplotype DQA1*01:01/DQB1*05:01/DRB1*01:01 occurred in 43.5% of HSP patients, whereas its frequency was 8.2% in IBD patients and 15.0% in the reference population. HSP patients with this haplotype showed similar baseline clinical findings and outcome as HSP patients negative for the haplotype. In IBD patients, no polymorphism or HLA allele appeared significant at the genome-wide level. CONCLUSIONS: Our results suggest that haplotype DQA1*01:01/DQB1*05:01/DRB1*01:01 is associated with susceptibility to HSP, but not with the severity of the kidney involvement. These HLA associations did not occur in IBD patients, suggesting that they are specific to HSP and not related to susceptibility to autoimmune diseases in general.

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. KEY POINTS: •Creation of pilus-secreting mutant (PA11) of the key probiotic LGG. •Strain PA11 is defective in a functional housekeeping sortase SrtA. •Strain PA11 opens novel biotherapeutic application avenues. Graphical abstract.

Post-copulatory genetic matchmaking: HLA-dependent effects of cervical mucus on human sperm function.

Several studies have demonstrated that women show pre-copulatory mating preferences for human leucocyte antigen (HLA)-dissimilar men. A fascinating, yet unexplored, possibility is that the ultimate mating bias towards HLA-dissimilar partners could occur after copulation, at the gamete level. Here, we explored this possibility by investigating whether the selection towards HLA-dissimilar partners occurs in the cervical mucus. After combining sperm and cervical mucus from multiple males and females (full factorial design), we found that sperm performance (swimming velocity, hyperactivation, and viability) was strongly influenced by the male-female combination. This indicates that sperm fertilization capability may be dependent on the compatibility between cervical mucus (female) and sperm (male). We also found that sperm viability was associated with partners' HLA dissimilarity, indicating that cervical mucus may selectively facilitate later gamete fusion between immunogenetically compatible partners. Together, these results provide novel insights into the female-mediated sperm selection (cryptic female choice) in humans and indicate that processes occurring after copulation may contribute to the mating bias towards HLA-dissimilar partners. Finally, by showing that sperm performance in cervical mucus is influenced by partners' genetic compatibility, the present findings may promote a deeper understanding of infertility.

Gamete-level immunogenetic incompatibility in humans-towards deeper understanding of fertilization and infertility?

Human leukocyte antigen (HLA) immune genes play an important role in partner selection, but it has remained unclear if nonrandom pairing with respect to parental HLA genes could occur at the level of the gametes. We tested this possibility by investigating whether the sperm fertilization competence in humans is dependent on HLA genotype combination of the partners. We conducted a full-factorial experiment, in which the sperm physiological preparation for fertilization among multiple males was studied in the presence of follicular fluid (oocyte surrounding bioactive liquid) of several females. All the studied sperm pre-fertilization physiological parameters (motility, hyperactivation, acrosome reaction, and viability) were strongly dependent on male-female combination. In other words, follicular fluids (women) that induce strong sperm physiological response in some males often induce much weaker response in the other(s). Sperm physiological responses were stronger in HLA-dissimilar male-female pairs than in HLA-similar combinations, but none of the measured sperm traits were associated with genome-wide similarity. Together, these findings shed new light on the evolutionary and immunological mechanisms of fertilization. Furthermore, our results raise an intriguing possibility that against currently prevailing WHO's definition, infertility may not represent exclusively a pathological condition, but may also result from immunogenetic incompatibility of the gametes.

Colonic Mucosal Microbiota and Association of Bacterial Taxa with the Expression of Host Antimicrobial Peptides in Pediatric Ulcerative Colitis.

Inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease (CD), are chronic debilitating disorders of unknown etiology. Over 200 genetic risk loci are associated with IBD, highlighting a key role for immunological and epithelial barrier functions. Environmental factors account for the growing incidence of IBD, and microbiota are considered as an important contributor. Microbiota dysbiosis can lead to a loss of tolerogenic immune effects and initiate or exacerbate inflammation. We aimed to study colonic mucosal microbiota and the expression of selected host genes in pediatric UC. We used high-throughput 16S rDNA sequencing to profile microbiota in colonic biopsies of pediatric UC patients (n = 26) and non-IBD controls (n = 27). The expression of 13 genes, including five for antimicrobial peptides, in parallel biopsies was assessed with qRT-PCR. The composition of microbiota between UC and non-IBD differed significantly (PCoA, p = 0.001). UC children had a decrease in Bacteroidetes and an increase in several family-level taxa including Peptostreptococcaceae and Enterobacteriaceae, which correlated negatively with the expression of antimicrobial peptides REG3G and DEFB1, respectively. Enterobacteriaceae correlated positively with the expression siderophore binding protein LCN2 and Betaproteobacteria negatively with DEFB4A expression. The results indicate that reciprocal interaction of epithelial microbiota and defense mechanisms play a role in UC.

Extended HLA Haplotypes and Their Impact on DPB1 Matching of Unrelated Hematologic Stem Cell Transplant Donors.

Although HLA-DPB1 has long been considered of lesser importance in the selection of an unrelated donor (URD) hematologic stem cell transplantation, currently in many instances the DPB1 type of the donor is relevant or even critical. At present, however, only a minority of registry donors are DPB1 typed. It is also unclear to what extent the DPB1 alleles are linked to the 5-locus HLA-A-, B-, C-, DRB1, -DQB1 haplotypes. We sought to study whether there is such a linkage by using donors in the Finnish Stem Cell Registry as the study population. The 6-locus HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 haplotype frequencies were estimated from a group of 43,365 Finnish registry donors using the German National Bone Marrow Registry algorithm. Five-locus haplotype (HLA-A, -B, -C, -DRB1, -DQB1) and HLA-DPB1 allele frequencies were calculated as marginal frequencies of the estimated 6-locus haplotype frequencies. The Finnish average frequency of individual DPB1 alleles was compared with their respective frequencies in association with individual 5-locus HLA haplotypes (haplotype-specific frequencies). Finally, the probability of DPB1 matching in 10/10 matched URD transplants was assessed. Haplotype-specific DPB1 frequencies differed significantly from the average DPB1 frequencies in 81 of 100 most frequent Finnish 5-locus HLA haplotypes, including some infrequent DPB1 alleles that were associated almost exclusively with certain individual 5-locus haplotypes. Five-locus haplotypes that are enriched in Finland but rare among other Europeans carried stronger DPB1 associations than haplotypes that are frequent European-wide. Finally, 10/10 matched transplants from domestic registry donors were significantly more likely to also be DPB1 matched than those from foreign donors. The results indicate an extension of linkage disequilibrium in the MHC complex in the Finnish population. With continuing upfront DPB1 typing of registry donors, it will be possible to perform similar extended 6-locus haplotype frequency estimations also in other registries. The associations are likely to be population specific but may be weaker in more heterogeneous populations. In the future the results might be used to predict the probability of DPB1 match or permissive/nonpermissive DPB1 mismatch for non-DPB1 typed donors in registry donor searches.

Increased MHC Matching by C4 Gene Compatibility in Unrelated Donor Hematopoietic Stem Cell Transplantation.

HLA matching is a prerequisite for successful allogeneic hematopoietic stem cell transplantation (HSCT) because it lowers the occurrence and severity of graft-versus-host disease (GVHD). However, matching a few alleles of the classic HLA genes only may not ensure matching of the entire MHC region. HLA haplotype matching has been reported to be beneficial in HSCT because of the variation relevant to GVHD risk in the non-HLA region. Because polymorphism in the MHC is highly population specific, we hypothesized that donors from the Finnish registry are more likely to be matched at a higher level for the Finnish patients than donors from other registries. In the present study we determined 25 single nucleotide polymorphisms (SNPs) of the complement component 4 (C4) gene in the γ-block segment of MHC from 115 Finnish HSCT patients and their Finnish (n = 201) and non-Finnish (n = 280) donor candidates. Full matching of HLA alleles and C4 SNPs, independently or additively, occurred more likely in the Finnish-Finnish group as compared with the Finnish-non-Finnish group (P < .003). This was most striking in cases with HLA haplotypes typical of the Finnish population. Patients with ancestral HLA haplotypes (AH) were more likely to find a full HLA and C4 matched donor, regardless of donor origin, as compared with patients without AH (P < .0001). Despite the clear differences at the population level, we could not find a statistical association between C4 matching and clinical outcome. The results suggest that screening C4 SNPs can be advantageous when an extended MHC matching or HLA haplotype matching in HSCT is required. This study also supports the need for small population-specific stem cell registries.

Romboutsia hominis sp. nov., the first human gut-derived representative of the genus Romboutsia, isolated from ileostoma effluent.

A Gram-stain-positive, motile, rod-shaped, obligately anaerobic bacterium, designated FRIFIT, was isolated from human ileostoma effluent and characterized. On the basis of 16S rRNA gene sequence similarity, strain FRIFIT was most closely related to the species Romboutsia ilealis CRIBT (97.7 %), Romboutsia lituseburensis DSM 797T (97.6 %) and Romboutsia sedimentorum LAM201T (96.6 %). The level of DNA-DNA relatedness between strain FRIFIT and R. ilealis CRIBT was 13.9±3.3 % based on DNA-DNA hybridization. Whole genome sequence-based average nucleotide identity between strain FRIFIT and closely related Romboutsia strains ranged from 78.4-79.1 %. The genomic DNA G+C content of strain FRIFIT was 27.8 mol%. The major cellular fatty acids of strain FRIFIT were saturated and unsaturated straight-chain C12-C19 fatty acids as well as cyclopropane fatty acids, with C16 : 0 being the predominant fatty acid. The polar lipid profile comprised five phospholipids and six glycolipids. These results, together with differences in phenotypic features, support the proposal that strain FRIFIT represents a novel species within the genus Romboutsia, for which the name Romboutsiahominis sp. nov. is proposed. The type strain is FRIFIT (=DSM 28814T=KCTC 15553T).

An Inducible Operon Is Involved in Inulin Utilization in Lactobacillus plantarum Strains, as Revealed by Comparative Proteogenomics and Metabolic Profiling.

The draft genomes of Lactobacillus plantarum strains isolated from Asian fermented foods, infant feces, and shrimp intestines were sequenced and compared to those of well-studied strains. Among 28 strains of L. plantarum, variations in the genomic features involved in ecological adaptation were elucidated. The genome sizes ranged from approximately 3.1 to 3.5 Mb, of which about 2,932 to 3,345 protein-coding sequences (CDS) were predicted. The food-derived isolates contained a higher number of carbohydrate metabolism-associated genes than those from infant feces. This observation correlated to their phenotypic carbohydrate metabolic profile, indicating their ability to metabolize the largest range of sugars. Surprisingly, two strains (P14 and P76) isolated from fermented fish utilized inulin. ß-Fructosidase, the inulin-degrading enzyme, was detected in the supernatants and cell wall extracts of both strains. No activity was observed in the cytoplasmic fraction, indicating that this key enzyme was either membrane-bound or extracellularly secreted. From genomic mining analysis, a predicted inulin operon of fosRABCDXE, which encodes ß-fructosidase and many fructose transporting proteins, was found within the genomes of strains P14 and P76. Moreover, pts1BCA genes, encoding sucrose-specific IIBCA components involved in sucrose transport, were also identified. The proteomic analysis revealed the mechanism and functional characteristic of the fosRABCDXE operon involved in the inulin utilization of L. plantarum The expression levels of the fos operon and pst genes were upregulated at mid-log phase. FosE and the LPXTG-motif cell wall anchored ß-fructosidase were induced to a high abundance when inulin was present as a carbon source. IMPORTANCE: Inulin is a long-chain carbohydrate that may act as a prebiotic, which provides many health benefits to the host by selectively stimulating the growth and activity of beneficial bacteria in the colon. While certain lactobacilli can catabolize inulin, this has not yet been described for Lactobacillus plantarum, and an associated putative inulin operon has not been reported in this species. By using comparative and functional genomics, we showed that two L. plantarum strains utilized inulin and identified functional inulin operons in their genomes. The proteogenomic data revealed that inulin degradation and uptake routes, which related to the fosRABCDXE operon and pstBCA genes, were widely expressed among L. plantarum strains. The present work provides a novel understanding of gene regulation and mechanisms of inulin utilization in probiotic L. plantarum generating opportunities for synbiotic product development.

Lactobacillus oligofermentans glucose, ribose and xylose transcriptomes show higher similarity between glucose and xylose catabolism-induced responses in the early exponential growth phase.

BACKGROUND: Lactobacillus oligofermentans has been mostly isolated from cold-stored packaged meat products in connection with their spoilage, but its precise role in meat spoilage is unknown. It belongs to the L. vaccinostercus group of obligate heterofermentative lactobacilli that generally ferment pentoses (e.g. xylose and ribose) more efficiently than hexoses (e.g. glucose). However, more efficient hexose utilization can be induced. The regulation mechanisms of the carbohydrate catabolism in such bacteria have been scarcely studied. To address this question, we provided the complete genome sequence of L. oligofermentans LMG 22743(T) and generated time course transcriptomes during its growth on glucose, ribose and xylose. RESULTS: The genome was manually annotated and its main functional features were examined. L. oligofermentans was confirmed to be able to efficiently utilize several hexoses and maltose, which is, presumably, induced by its repeated cultivation with glucose in vitro. Unexpectedly, in the beginning of the exponential growth phase, glucose- and xylose-induced transcriptome responses were more similar, whereas toward the end of the growth phase xylose and ribose transcriptomes became more alike. The promoter regions of genes simultaneously upregulated both on glucose and xylose in comparison with ribose (particularly, hexose and xylose utilization genes) were found to be enriched in the CcpA- binding site. Transcriptionally, no glucose-induced carbon catabolite repression was detected. The catabolism of glucose, which requires initial oxidation, led to significant overexpression of the NAD(P)H re-oxidation genes, the upstream regions of which were found to contain a motif, which was highly similar to a Rex repressor binding site. CONCLUSIONS: This paper presents the second complete genome and the first study of carbohydrate catabolism-dependent transcriptome response for a member of the L. vaccinostercus group. The transcriptomic changes detected in L. oligofermentans for growth with different carbohydrates differ significantly from those of facultative heterofermentative lactobacilli. The mechanism of CcpA regulation, putatively contributing to the observed similarities between glucose- and xylose-induced transcriptome responses and the absence of stringent carbon catabolite control, requires further studies. Finally, the cell redox balance maintenance, in terms of the NAD(P)+/NAD(P)H ratio, was predicted to be regulated by the Rex transcriptional regulator, supporting the previously made inference of Rex-regulons for members of the Lactobacillaceae family.

Polymorphisms, Chromosomal Rearrangements, and Mutator Phenotype Development during Experimental Evolution of Lactobacillus rhamnosus GG.

UNLABELLED: Lactobacillus rhamnosus GG is a lactic acid bacterium widely marketed by the food industry. Its genomic analysis led to the identification of a gene cluster encoding mucus-binding SpaCBA pili, which is located in a genomic island enriched in insertion sequence (IS) elements. In the present study, we analyzed by genome-wide resequencing the genomic integrity of L. rhamnosus GG in four distinct evolutionary experiments conducted for approximately 1,000 generations under conditions of no stress or salt, bile, and repetitive-shearing stress. Under both stress-free and salt-induced stress conditions, the GG population (excluding the mutator lineage in the stress-free series [see below]) accumulated only a few single nucleotide polymorphisms (SNPs) and no frequent chromosomal rearrangements. In contrast, in the presence of bile salts or repetitive shearing stress, some IS elements were found to be activated, resulting in the deletion of large chromosomal segments that include the spaCBA-srtC1 pilus gene cluster. Remarkably, a high number of SNPs were found in three strains obtained after 900 generations of stress-free growth. Detailed analysis showed that these three strains derived from a founder mutant with an altered DNA polymerase subunit that resulted in a mutator phenotype. The present work confirms the stability of the pilus production phenotype in L. rhamnosus GG under stress-free conditions, highlights the possible evolutionary scenarios that may occur when this probiotic strain is extensively cultured, and identifies external factors that affect the chromosomal integrity of GG. The results provide mechanistic insights into the stability of GG in regard to its extensive use in probiotic and other functional food products. IMPORTANCE: Lactobacillus rhamnosus GG is a widely marketed probiotic strain that has been used in numerous clinical studies to assess its health-promoting properties. Hence, the stability of the probiotic functions of L. rhamnosus GG is of importance, and here we studied the impact of external stresses on the genomic integrity of L. rhamnosus GG. We studied three different stresses that are relevant for understanding its robustness and integrity under both ex vivo conditions, i.e., industrial manufacturing conditions, and in vivo conditions, i.e., intestinal tract-associated stress. Overall, our findings contribute to predicting the genomic stability of L. rhamnosus GG and its ecological performance.

Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG.

Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects.

Effects of bowel cleansing on the intestinal microbiota.

OBJECTIVE: An adequate bowel cleansing is essential for a successful colonoscopy. Although purgative consumption is safe for the patient, there is little consensus on how the intestinal microbiota is affected by the procedure, especially regarding the potential long-term consequences. DESIGN: 23 healthy subjects were randomised into two study groups consuming a bowel preparation (Moviprep), either in two separate doses of 1 L or as a single 2-L dose. Participants donated faecal samples at the baseline, after bowel cleansing, 14 and 28 days after the treatment. The intestinal microbiota composition was determined with phylogenetic microarray as well as quantitative PCR analysis and correlated with the previously quantified faecal serine proteases. RESULTS: The lavage introduced an instant and substantial change to the intestinal microbiota. The total microbial load was decreased by 31-fold and 22% of the participants lost the subject-specificity of their microbiota. While the bacterial levels and community composition were essentially restored within 14 days, the rate of recovery was dose dependent: consumption of the purgative in a single dose had a more severe effect on the microbiota composition than that of a double dose, and notably increased the levels of Proteobacteria, Fusobacteria and bacteria related to Dorea formicigenerans. The abundance of the latter also correlated with the amount of faecal serine proteases that were increased after purging. CONCLUSIONS: Our results suggest that the bowel cleansing using two separate dosages introduces fewer alterations to the intestinal microbiota than a single dose and hence may be preferred in clinical practice.

Colonic metaproteomic signatures of active bacteria and the host in obesity.

Obesity is associated with the intestinal microbiota in humans but the underlying mechanisms are yet to be fully understood. Our previous phylogenetic study showed that the faecal microbiota profiles of nonobese versus obese and morbidly obese individuals differed. Here, we have extended this analysis with a characterization of the faecal metaproteome, in order to detect differences at a functional level. Proteins were extracted from crude faecal samples of 29 subjects, separated by 1D gel electrophoresis and characterized using RP LC-MS/MS. The peptide data were analyzed in database searches with two complementary algorithms, OMSSA and X!Tandem, to increase the number of identifications. Evolutionary genealogy of genes: nonsupervised orthologous groups (EggNOG) database searches resulted in the functional annotation of over 90% of the identified microbial and human proteins. Based on both bacterial and human proteins, a clear clustering of obese and nonobese samples was obtained that exceeded the phylogenetic separation in dimension. Moreover, integration of the metaproteomics and phylogenetic datasets revealed notably that the phylum Bacteroidetes was metabolically more active in the obese than nonobese subjects. Finally, significant correlations between clinical measurements and bacterial gene functions were identified. This study emphasizes the importance of integrating data of the host and microbiota to understand their interactions.

Improved taxonomic assignment of human intestinal 16S rRNA sequences by a dedicated reference database.

BACKGROUND: Current sequencing technology enables taxonomic profiling of microbial ecosystems at high resolution and depth by using the 16S rRNA gene as a phylogenetic marker. Taxonomic assignation of newly acquired data is based on sequence comparisons with comprehensive reference databases to find consensus taxonomy for representative sequences. Nevertheless, even with well-characterised ecosystems like the human intestinal microbiota it is challenging to assign genus and species level taxonomy to 16S rRNA amplicon reads. A part of the explanation may lie in the sheer size of the search space where competition from a multitude of highly similar sequences may not allow reliable assignation at low taxonomic levels. However, when studying a particular environment such as the human intestine, it can be argued that a reference database comprising only sequences that are native to the environment would be sufficient, effectively reducing the search space. RESULTS: We constructed a 16S rRNA gene database based on high-quality sequences specific for human intestinal microbiota, resulting in curated data set consisting of 2473 unique prokaryotic species-like groups and their taxonomic lineages, and compared its performance against the Greengenes and Silva databases. The results showed that regardless of used assignment algorithm, our database improved taxonomic assignation of 16S rRNA sequencing data by enabling significantly higher species and genus level assignation rate while preserving taxonomic diversity and demanding less computational resources. CONCLUSION: The curated human intestinal 16S rRNA gene taxonomic database of about 2500 species-like groups described here provides a practical solution for significantly improved taxonomic assignment for phylogenetic studies of the human intestinal microbiota.