Mucosal Niche Research Articles

Lactobacillus gasseri requires peptides, not proteins or free amino acids, for growth in milk

Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious peptides to enteric lactobacilli including L. gasseri after peptic digestion in the gastrointestinal tract. This is the first report showing peptide requirement of L. gasseri and efficacy of pepsinolysis on the growth of L. gasseri and its relatives in milk. This study would contribute to increasing usability of L. gasseri and its relatives as probiotics in dairy foods.

Staphylococcus aureus manganese transport protein C (MntC) is an extracellular matrix- and plasminogen-binding protein.

Infections caused by Staphylococcus aureus – particularly nosocomial infections - represent a great concern. Usually, the early stage of pathogenesis consists on asymptomatic nasopharynx colonization, which could result in dissemination to other mucosal niches or invasion of sterile sites, such as blood. This pathogenic route depends on scavenging of nutrients as well as binding to and disrupting extracellular matrix (ECM). Manganese transport protein C (MntC), a conserved manganese-binding protein, takes part in this infectious scenario as an ion-scavenging factor and surprisingly as an ECM and coagulation cascade binding protein, as revealed in this work. This study showed a marked ability of MntC to bind to several ECM and coagulation cascade components, including laminin, collagen type IV, cellular and plasma fibronectin, plasminogen and fibrinogen by ELISA. The MntC binding to plasminogen appears to be related to the presence of surface-exposed lysines, since previous incubation with an analogue of lysine residue, ε-aminocaproic acid, or increasing ionic strength affected the interaction between MntC and plasminogen. MntC-bound plasminogen was converted to active plasmin in the presence of urokinase plasminogen activator (uPA). The newly released plasmin, in turn, acted in the cleavage of the α and β chains of fibrinogen. In conclusion, we describe a novel function for MntC that may help staphylococcal mucosal colonization and establishment of invasive disease, through the interaction with ECM and coagulation cascade host proteins. These data suggest that this potential virulence factor could be an adequate candidate to compose an anti-staphylococcal human vaccine formulation.

Genomic and phenotypic evidence for probiotic influences ofLactobacillus gasserion human health

Certain lactic acid bacteria (LAB) have the capacity to occupy mucosal niches of humans, including the oral cavity, gastrointestinal tract, and vagina. Among commensal, LAB are species of the acidophilus complex, which have proven to be a substantial reservoir for microorganisms with probiotic attributes. Specifically, Lactobacillus gasseri is an autochthonous microorganism which has been evaluated for probiotic activity based on the availability of genome sequence and species-specific adaptation to the human mucosa. Niche-related characteristics of L. gasseri contributing to indigenous colonization include tolerance of low pH environments, resistance to bile salts, and adhesion to the host epithelium. In humans, L. gasseri elicits various health benefits through its antimicrobial activity, bacteriocin production, and immunomodulation of the innate and adaptive systems. The genomic and empirical evidence supporting use of L. gasseri in probiotic applications is substantiated by clinical trial data displaying maintenance of vaginal homeostasis, mitigation of Helicobacter pylori infection, and amelioration of diarrhea.

Noncanonical dendritic cell differentiation and survival driven by a bacteremic pathogen

Maintenance of blood DC homeostasis is essential to preventing autoimmunity while controlling chronic infection. However, the ability of bacteremic pathogens to directly regulate blood DC homeostasis has not been defined. One such bacteremic pathogen, Porphyromonas gingivalis, is shown by our group to survive within mDCs under aerobic conditions and therein, metastasize from its oral mucosal niche. This is accompanied by expansion of the blood mDC pool in vivo, independently of canonical DC poietins. We presently know little of how this bacteremic pathogen causes blood DC expansion and the pathophysiological significance. This work shows that optimum differentiation of MoDCs from primary human monocytes, with or without GM-CSF/IL-4, is dependent on infection with P. gingivalis strains expressing the DC-SIGN ligand mfa-1. DC differentiation is lost when DC-SIGN is blocked with its ligand HIV gp120 or knocked out by siRNA gene silencing. Thus, we have identified a novel, noncanonical pathway of DC differentiation. We term these PDDCs and show that PDDCs are bona fide DCs, based on phenotype and phagocytic activity when immature and the ability to up-regulate accessory molecules and stimulate allo-CD4(+) T cell proliferation when matured. The latter is dependent on the P. gingivalis strain used to initially "educate" PDDCs. Moreover, we show that P. gingivalis-infected, conventional MoDCs become resistant to apoptosis and inflammatory pyroptosis, as determined by levels of Annexin V and caspase-8, -3/7, and -1. Taken together, we provide new insights into how a relatively asymptomatic bacteremia may influence immune homeostasis and promote chronic inflammation.

Su1266 In Vitro Generated Regulatory T Cells From Blood Suppress Mucosal Effector T Cells in Crohn's Disease Patients

Background: Thymically derived CD4+CD25hiCD127loFOXP3+ regulatory T cells (Tregs) maintain immunological tolerance and prevent inappropriate activation of multiple immune cell types. Quantitative and qualitative Treg defects are seen in inflamed Crohn's (CD) mucosa, suggesting that insufficient Treg-mediated suppressor function contributes to chronic mucosal inflammation in CD. Consequently, cell-based therapy with autologous Tregs to restore mucosal Treg numbers and function is conceptually attractive. The recent use of in vitro expanded umbilical cord blood (UCB) Tregs to prevent GvHD following UCB transplantation confirmed the safety of this approach in humans. The optimal method of generating Tregs in vitro from CD patients to produce a homogenous, phenotypically stable yet functionally suppressive population on expansion is unknown, as is the effect of these cells on target organs: mesenteric lymph node (MLN) and lamina propria (LP) immune cells in inflamed CD gut. Aim: To define the optimal method of GMP-compatible Treg isolation and expansion from CD patients' blood with reference to homogeneity, phenotypic stability and suppressive ability of the cell product. Results: Tregs were expanded for 2028 days in X-VIVO15 with 5% human AB serum, IL-2 and Rapamycin. Tregs initially isolated using GMP-compatible MACS-based enrichment had unacceptably high CD8+ contamination (.20%) and high expression of IL-17 and IFNγ (n=5 independent experiments). CD4+CD25+CD127lo Tregs were then sorted from CD blood on the basis of CD45RA expression and expanded a median of 127-fold (IQR 58-531) to yield a .97% CD4+CD25hiCD127loFOXP3+ population (n=10). Expanded CD45RA+ Tregs expressed functionally relevant markers including CTLA-4, ICOS, CD27 and CD62L ( .97% each) and TGF-β but not IL-10. CD45RA+ Tregs expressed ,1% IL-17 by FACS and had greater phenotypic stability than expanded CD45RATregs, with less spontaneous (p,0.05) and induced (p,0.01) IL-17 production by ELISA. CD45RA+ Tregs highly suppressed autologous CD4+CD25proliferation at 96h (median=97.3% [96.6%-99.1%] at 1:1 ratio); and were more effective than CD45RATregs at suppressing CD4+CD25activation (p=0.003), assessed by CD154 expression at 7h. Importantly, using CD3+ T responders from inflamed Crohn's resections, CD45RA+ Tregs suppressed MLN CD3+ proliferation (88.4% [53.8-93.2%]), MLN CD154 expression (82.9% [60.1-87.6%]) and LP CD3+ CD154 expression (64.3% [64.377 . 9%] ) i n a do s e d ep enden t manne r . Conc lu s i ons : FACS so r t i n g o f a CD4+CD25hiCD127loCD45RA+ precursor population is required to produce a homogenous, phenotypically stable in vitro expanded cell product from CD patients. For the first time, we show that in vitro expanded Tregs suppress immune cell activation in relevant mucosal immune niches, strengthening the rationale for clinical trials of autologous in vitro expanded Tregs in CD.

Methanogenesis in Irritable Bowel Syndrome: A Lot of Hot Air?

The human gut consists of several thousand species ofprokaryotes including bacteria and archaea that havemultiple metabolic and constitutive effects. A large pro-portion of these species are autochthonous, i.e., bacteriathat are present among the gut mucosal niches that haveevolved to survive in this ecological space while presum-ably providing a beneficial effect to the host [1]. Changesin this flora have been associated with inflammatory boweldisease, cirrhosis, and irritable bowel syndrome (IBS)[2–4]. In most disease states, there is a relative increase inpotentially pathogenic bacteria compared to healthy hostsalong with a reduction in the overall bio-diversity.A stumbling block in the characterization of the gutmicrobiome is the inability of the majority of species,especially anaerobic species, to be grown using standardculture methods [5]. For example, it has been suggestedthat less than 1 % of an environmental community can becultured using typical growth media [6]. Initially, cloningand sequencing was performed to elucidate the communityin the human gut but this was both tedious and expensive[7]. The introduction of NextGen sequencing technologyhas revolutionized the field of microbial ecology andallows for the rapid in-depth analysis of many differentenvironmental samples. To put this into perspective, thetypical fluorescent sequencer will produce 96 reads per daywhile the NextGen Roche GS-FLX will produce 500,000reads per day. Thus, the new technology produces 20 yearsworth of cloning and sequencing in an afternoon. Fur-thermore, multitag pyrosequencing (MTPS) has become apowerful and routine method for microbial communityanalysis [8–11] that takes advantage of this high-through-put technology. This methodology essentially barcodessamples that are then mixed and sequenced on one high-throughput sequencing run and then the reads are sortedinto their corresponding samples based on the barcode.Specific amplification primers can be used to selectivelyamplify variable regions of the 16S rRNA of archaea orbacteria to enumerate these components in the gutmicrobiome. Therefore, the profusion of recent studies thatevaluate the culture-independent methods of fecal bacterialenumeration opens an interesting chapter in our under-standing of this complex system.It has been known for decades that adult humans excretemethane and approximately one-third of all Americansproduce detectable methane [12, 13]. Methane is producedin the human colon by methanogenic archaea by reducingCO

Host sialoglycans and bacterial sialidases: a mucosal perspective

Sialic acids are nine-carbon-backbone sugars that occupy outermost positions on vertebrate cells and secreted sialoglycoproteins. These negatively charged hydrophilic carbohydrates have a variety of biological, biophysical and immunological functions. Mucosal surfaces and secretions of the mouth, airway, gut and vagina are especially sialoglycan-rich. Given their prominent positions and important functions, a variety of microbial strategies have targeted host sialic acids for adherence, mimicry and/or degradation. Here we review the roles of bacterial sialidases (neuraminidases) during colonization and pathogenesis of mammalian mucosal surfaces. Evidence is presented to support the myriad roles of mucosal sialoglycans in protecting the host from bacterial infection. In opposition, many bacteria hydrolyse sialic acids during associations with the gastrointestinal, oral, respiratory and reproductive tracts. Sialidases promote bacterial survival in mucosal niche environments in several ways, including: (i) nutritional benefits of sialic acid catabolism, (ii) unmasking of cryptic host ligands used for adherence, (iii) participation in biofilm formation and (iv) modulation of immune function. Bacterial sialidases are among the best-studied enzymes involved in pathogenesis and may also drive commensal and/or symbiotic host associations. Future studies should continue to define host substrates of bacterial sialidases and the mechanisms of their pathologic, commensal and symbiotic interactions with the mammalian host.

Helicobacter pylori CagA Triggers Expression of the Bactericidal Lectin REG3γ via Gastric STAT3 Activation

BackgroundMost of what is known about the Helicobacter pylori (H. pylori) cytotoxin, CagA, pertains to a much-vaunted role as a determinant of gastric inflammation and cancer. Little attention has been devoted to potential roles of CagA in the majority of H. pylori infected individuals not showing oncogenic progression, particularly in relation to host tolerance. Regenerating islet-derived (REG)3γ encodes a secreted C-type lectin that exerts direct bactericidal activity against Gram-positive bacteria in the intestine. Here, we extend this paradigm of lectin-mediated innate immunity, showing that REG3γ expression is triggered by CagA in the H. pylori-infected stomach.Methodology/Principal FindingsIn human gastric mucosal tissues, REG3γ expression was significantly increased in CagA-positive, compared to CagA-negative H. pylori infected individuals. Using transfected CagA-inducible gastric MKN28 cells, we recapitulated REG3γ induction in vitro, also showing that tyrosine phosphorylated, not unphosphorylated CagA triggers REG3γ transcription. In concert with induced REG3γ, pro-inflammatory signalling downstream of the gp130 cytokine co-receptor via the signal transducer and activator of transcription (STAT)3 and transcription of two cognate ligands, interleukin(IL)-11 and IL-6, were significantly increased. Exogenous IL-11, but not IL-6, directly stimulated STAT3 activation and REG3γ transcription. STAT3 siRNA knockdown or IL-11 receptor blockade respectively abrogated or subdued CagA-dependent REG3γ mRNA induction, thus demonstrating a requirement for uncompromised signalling via the IL-11/STAT3 pathway. Inhibition of the gp130-related SHP2-(Ras)-ERK pathway did not affect CagA-dependent REG3γ induction, but strengthened STAT3 activation as well as augmenting transcription of mucosal innate immune regulators, IL-6, IL-8 and interferon-response factor (IRF)1.Conclusions/SignificanceOur results support a model of CagA-directed REG3γ expression in gastric epithelial cells via activation of the IL-11/gp130/STAT3 pathway. This response might allow Gram-negative H. pylori to manipulate host immunity to favour its own survival, by reducing the fitness of co-habiting Gram-positive bacteria with which it competes for resources in the gastric mucosal niche.

Attenuated CagA Oncoprotein in Helicobacter pylori from Amerindians in Peruvian Amazon

Population genetic analyses of bacterial genes whose products interact with host tissues can give new understanding of infection and disease processes. Here we show that strains of the genetically diverse gastric pathogen Helicobacter pylori from Amerindians from the remote Peruvian Amazon contain novel alleles of cagA, a major virulence gene, and reveal distinctive properties of their encoded CagA proteins. CagA is injected into the gastric epithelium where it hijacks pleiotropic signaling pathways, helps Hp exploit its special gastric mucosal niche, and affects the risk that infection will result in overt gastroduodenal diseases including gastric cancer. The Amerindian CagA proteins contain unusual but functional tyrosine phosphorylation motifs and attenuated CRPIA motifs, which affect gastric epithelial proliferation, inflammation, and bacterial pathogenesis. Amerindian CagA proteins induced less production of IL-8 and cancer-associated Mucin 2 than did those of prototype Western or East Asian strains and behaved as dominant negative inhibitors of action of prototype CagA during mixed infection of Mongolian gerbils. We suggest that Amerindian cagA is of relatively low virulence, that this may have been selected in ancestral strains during infection of the people who migrated from Asia into the Americas many thousands of years ago, and that such attenuated CagA proteins could be useful therapeutically.

Molecular analysis of the luminal- and mucosal-associated intestinal microbiota in diarrhea-predominant irritable bowel syndrome

Alterations in the intestinal microbiota have been suggested as an etiological factor in the pathogenesis of irritable bowel syndrome (IBS). This study used a molecular fingerprinting technique to compare the composition and biodiversity of the microbiota within fecal and mucosal niches between patients with diarrhea-predominant IBS (D-IBS) and healthy controls. Terminal-restriction fragment (T-RF) length polymorphism (T-RFLP) fingerprinting of the bacterial 16S rRNA gene was used to perform microbial community composition analyses on fecal and mucosal samples from patients with D-IBS (n = 16) and healthy controls (n = 21). Molecular fingerprinting of the microbiota from fecal and colonic mucosal samples revealed differences in the contribution of T-RFs to the microbiota between D-IBS patients and healthy controls. Further analysis revealed a significantly lower (1.2-fold) biodiversity of microbes within fecal samples from D-IBS patients than healthy controls (P = 0.008). No difference in biodiversity in mucosal samples was detected between D-IBS patients and healthy controls. Multivariate analysis of T-RFLP profiles demonstrated distinct microbial communities between luminal and mucosal niches in all samples. Our findings of compositional differences in the luminal- and mucosal-associated microbiota between D-IBS patients and healthy controls and diminished microbial biodiversity in D-IBS fecal samples further support the hypothesis that alterations in the intestinal microbiota may have an etiological role in the pathogenesis of D-IBS and suggest that luminal and mucosal niches need to be investigated.

The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota.

Mucosal surfaces constantly encounter microbes. Toll-like receptors (TLRs) mediate recognition of microbial patterns to eliminate pathogens. By contrast, we demonstrate that the prominent gut commensal Bacteroides fragilis activates the TLR pathway to establish host-microbial symbiosis. TLR2 on CD4(+) T cells is required for B. fragilis colonization of a unique mucosal niche in mice during homeostasis. A symbiosis factor (PSA, polysaccharide A) of B. fragilis signals through TLR2 directly on Foxp3(+) regulatory T cells to promote immunologic tolerance. B. fragilis lacking PSA is unable to restrain T helper 17 cell responses and is defective in niche-specific mucosal colonization. Therefore, commensal bacteria exploit the TLR pathway to actively suppress immunity. We propose that the immune system can discriminate between pathogens and the microbiota through recognition of symbiotic bacterial molecules in a process that engenders commensal colonization.

Surface Properties of Helicobacter pylori Urease Complex Are Essential for Persistence

The enzymatic activity of Helicobacter pylori's urease neutralises stomach acidity, thereby promoting infection by this pathogen. Urease protein has also been found to interact with host cells in vitro, although this property's possible functional importance has not been studied in vivo. To test for a role of the urease surface in the host/pathogen interaction, surface exposed loops that display high thermal mobility were targeted for inframe insertion mutagenesis. H. pylori expressing urease with insertions at four of eight sites tested retained urease activity, which in three cases was at least as stable as was wild-type urease at pH 3. Bacteria expressing one of these four mutant ureases, however, failed to colonise mice for even two weeks, and a second had reduced bacterial titres after longer term (3 to 6 months) colonisation. These results indicate that a discrete surface of the urease complex is important for H. pylori persistence during gastric colonisation. We propose that this surface interacts directly with host components important for the host-pathogen interaction, immune modulation or other actions that underlie H. pylori persistence in its special gastric mucosal niche.

The Candida albicans ESCRT Pathway Makes Rim101-Dependent and -Independent Contributions to Pathogenesis

Candida albicans is an opportunistic pathogen that colonizes diverse mucosal niches with distinct environmental characteristics. To adapt to these different sites, C. albicans must activate and attenuate a variety of signal transduction pathways. A mechanism of signal attenuation is through receptor endocytosis and subsequent vacuolar degradation, which requires the endosomal sorting complex required for transport (ESCRT) pathway. This pathway comprises several polyprotein complexes (ESCRT-0, -I, -II, -III, and -DS) that are sequentially recruited to the endosomal membrane. The ESCRT pathway also activates the Rim101 transcription factor, which governs expression of genes required for virulence. Here, we tested the hypothesis that the ESCRT pathway plays a Rim101-independent role(s) in pathogenesis. We generated deletion mutants in each ESCRT complex and determined that ESCRT-I, -II, and -III are required for Rim101 activation but that ESCRT-0 and ESCRT-DS are not. We found that the ESCRT-0 member Vps27 and ESCRT-DS components are required to promote epithelial cell damage and, using a murine model of oral candidiasis, found that the vps27Delta/Delta mutant had a decreased fungal burden compared to that of the wild type. We found that a high-dose inoculum can compensate for fungal burden defects but that mice colonized with the vps27Delta/Delta strain exhibit less morbidity than do mice infected with the wild-type strain. These results demonstrate that the ESCRT pathway has Rim101-independent functions for C. albicans virulence.

Abstract 3453: Phylogenetic fingerprinting of the fecal microbiota in colorectal cancer patients

Abstract Colonic adenomatous polyps are common tumors occurring in ∼50% of Western populations with ∼10% risk of malignant progression. To date, the environmental exposures proposed to promote the development of colorectal cancer (CRC) have been primarily dietary agents. However, the local environment to which the colonic mucosa is exposed is created by the bacterial microbiota of the colon and their metabolic products that include toxins. A member of the Bacteroidetes division, Bacteroides fragilis is a common human commensal, occupying a mucosal niche in the colon. One molecular class of B. fragilis, enterotoxigenic B. fragilis (ETBF) induces polyp formation in Min (multiple intestinal neopplasia) mice through a STAT3 pathway. Here we analyzed fecal DNA for gut flora composition in African Americans (n=12) with colon adenoma. A 16S rDNA cloning/sequencing experiment revealed that patients with colon adenomas have a higher rate of Bacteroides bacteria than healthy ones. These data was further confirmed using a HITChip (Human Intestinal Tract Chip) where samples from adenoma patients tended to cluster together pointing to common bacterial profiles. STAT3 induced in adenoma and tumor by IHC, indicating, signal activation perhaps through ETBF like bacteria. In conclusion, High throughput and sequencing approaches may help to better detect gut flora components including Bacteroides fragilis that induces colorectal carcinogenesis. In addition, the STAT3 expression results would confirm possible ETBF involvement on colon cancer risk via activating STAT3 downstream factor and molecules that are known to play a key role in the regulation of epithelial proliferation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3453.

Expression of foreign proteins on gram-positive commensal bacteria for mucosal vaccine delivery

Non-pathogenic Gram-positive oral commensal bacteria expressing recombinant fusion proteins on their cell surface have been successfully used to raise both a mucosal and a systemic immune response to foreign antigens while colonizing the oropharynx. In this system, fusion-protein vaccines are delivered and anchored to the surface of a commensal, which occupies the mucosal niche invaded by a particular pathogen. Surface expression of these foreign proteins is achieved by exploiting the common mechanism employed by Gram-positive bacteria for translocating and anchoring proteins to the cell surface. The process offers a safe alternative to the use of engineered pathogens as live vaccine delivery vehicles.