scholarly journals Adaptation of Akkermansia muciniphila to the Oxic-Anoxic Interface of the Mucus Layer

2016 ◽  
Vol 82 (23) ◽  
pp. 6983-6993 ◽  
Author(s):  
Janneke P. Ouwerkerk ◽  
Kees C. H. van der Ark ◽  
Mark Davids ◽  
Nico J. Claassens ◽  
Teresa Robert Finestra ◽  
...  
Keyword(s):  
Growth Enhancement ◽  
Strictly Anaerobic ◽  
Mucus Layer ◽  
Low Oxygen ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Akkermansia Muciniphila ◽  
Initial Oxygen ◽  
Low Levels

ABSTRACTAkkermansia muciniphilacolonizes the mucus layer of the gastrointestinal tract, where the organism can be exposed to the oxygen that diffuses from epithelial cells. To understand howA. muciniphilais able to survive and grow at this oxic-anoxic interface, its oxygen tolerance and response and reduction capacities were studied.A. muciniphilawas found to be oxygen tolerant. On top of this, under aerated conditions,A. muciniphilashowed significant oxygen reduction capacities and its growth rate and yield were increased compared to those seen under strict anaerobic conditions. Transcriptome analysis revealed an initial oxygen stress response upon exposure to oxygen. Thereafter, genes related to respiration were expressed, including those coding for the cytochromebdcomplex, which can function as a terminal oxidase. The functionality ofA. muciniphilacytochromebdgenes was proven by successfully complementing cytochrome-deficientEscherichia colistrain ECOM4. We conclude thatA. muciniphilacan use oxygen when it is present at nanomolar concentrations.IMPORTANCEThis article explains howAkkermansia muciniphila, previously described as a strictly anaerobic bacterium, is able to tolerate and even benefit from low levels of oxygen. Interestingly, we measured growth enhancement ofA. muciniphilaand changes in metabolism as a result of the oxygen exposure. In this article, we discuss similarities and differences of this oxygen-responsive mechanism with respect to those of other intestinal anaerobic isolates. Taken together, we think that these are valuable data that indicate how anaerobic intestinal colonizing bacteria can exploit low levels of oxygen present in the mucus layer and that our results have direct relevance for applicability, as addition of low oxygen concentrations could benefit thein vitrogrowth of certain anaerobic organisms.

scholarly journals How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Nicolas Kint ◽  
Carolina Alves Feliciano ◽  
Maria C. Martins ◽  
Claire Morvan ◽  
Susana F. Fernandes ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Anaerobic Bacteria ◽  
Growth Defect ◽  
Strictly Anaerobic ◽  
Low Oxygen ◽  
Air Exposure ◽  
Vegetative Cells ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

scholarly journals Evaluation of Immunostimulatory Activities of Synthetic Mannose-Containing Structures Mimicking the β-(1→2)-Linked Cell Wall Mannans of Candida albicans

2012 ◽  
Vol 19 (11) ◽  
pp. 1889-1893 ◽  
Author(s):  
Kaarina Ranta ◽  
Kaisa Nieminen ◽  
Filip S. Ekholm ◽  
Moniká Poláková ◽  
Mattias U. Roslund ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Peripheral Blood Mononuclear ◽  
Content Type ◽  
Mouse Macrophages ◽  
Ifn Γ ◽  
Low Levels ◽  
Necrosis Factor

ABSTRACTImmunostimulatory properties of synthetic structures mimicking the β-(1→2)-linked mannans ofCandida albicanswere evaluatedin vitro. Contrary to earlier observations, tumor necrosis factor (TNF) production was not detected after stimulation with mannotetraose in mouse macrophages. Divalent disaccharide 1,4-bis(α-d-mannopyranosyloxy)butane induced TNF and some molecules induced low levels of gamma interferon (IFN-γ) in human peripheral blood mononuclear cells (PBMC).

scholarly journals Redesigning Escherichia coli Metabolism for Anaerobic Production of Isobutanol

2011 ◽  
Vol 77 (14) ◽  
pp. 4894-4904 ◽  
Author(s):  
Cong T. Trinh ◽  
Johnny Li ◽  
Harvey W. Blanch ◽  
Douglas S. Clark
Keyword(s):  
Escherichia Coli ◽  
Anaerobic Growth ◽  
Mode Analysis ◽  
Glycolytic Flux ◽  
Strictly Anaerobic ◽  
Anaerobic Conditions ◽  
Content Type ◽  
E Coli ◽  
Model Predictions ◽  
Chromosomal Genes

ABSTRACTFermentation enables the production of reduced metabolites, such as the biofuels ethanol and butanol, from fermentable sugars. This work demonstrates a general approach for designing and constructing a production host that uses a heterologous pathway as an obligately fermentative pathway to produce reduced metabolites, specifically, the biofuel isobutanol. Elementary mode analysis was applied to design anEscherichia colistrain optimized for isobutanol production under strictly anaerobic conditions. The central metabolism ofE. coliwas decomposed into 38,219 functional, unique, and elementary modes (EMs). The model predictions revealed that during anaerobic growthE. colicannot produce isobutanol as the sole fermentative product. By deleting 7 chromosomal genes, the total 38,219 EMs were constrained to 12 EMs, 6 of which can produce high yields of isobutanol in a range from 0.29 to 0.41 g isobutanol/g glucose under anaerobic conditions. The remaining 6 EMs rely primarily on the pyruvate dehydrogenase enzyme complex (PDHC) and are typically inhibited under anaerobic conditions. The redesignedE. colistrain was constrained to employ the anaerobic isobutanol pathways through deletion of 7 chromosomal genes, addition of 2 heterologous genes, and overexpression of 5 genes. Here we present the design, construction, and characterization of an isobutanol-producingE. colistrain to illustrate the approach. The model predictions are evaluated in relation to experimental data and strategies proposed to improve anaerobic isobutanol production. We also show that the endogenous alcohol/aldehyde dehydrogenase AdhE is the key enzyme responsible for the production of isobutanol and ethanol under anaerobic conditions. The glycolytic flux can be controlled to regulate the ratio of isobutanol to ethanol production.

scholarly journals The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli

2017 ◽  
Vol 199 (16) ◽  
Author(s):  
Brian M. Meehan ◽  
Cristina Landeta ◽  
Dana Boyd ◽  
Jonathan Beckwith
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bond ◽  
Bond Formation ◽  
Anaerobic Growth ◽  
Strictly Anaerobic ◽  
Disulfide Bond Formation ◽  
Anaerobic Conditions ◽  
Content Type ◽  
E Coli ◽  
Laboratory Conditions

ABSTRACT Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli, intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restored by suppressor mutations in the disulfide bond isomerization system. Overall, our results underscore the importance of proper disulfide bond formation to pathways critical to E. coli viability under conditions where oxygen is limited. IMPORTANCE While the disulfide bond formation (DSB) system of E. coli has been studied for decades and has been shown to play an important role in the proper folding of many proteins, including some associated with virulence, it was considered dispensable for growth under most laboratory conditions. This work represents the first attempt to study the effects of the DSB system under strictly anaerobic conditions, simulating the environment encountered by pathogenic E. coli strains in the human intestinal tract. By demonstrating that the DSB system is essential for growth under such conditions, this work suggests that compounds inhibiting Dsb enzymes might act not only as antivirulents but also as true antibiotics.

scholarly journals Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori

2004 ◽  
Vol 53 (11) ◽  
pp. 1123-1128 ◽  
Author(s):  
Monique M Gerrits ◽  
Egbert-Jan van der Wouden ◽  
Dorine A Bax ◽  
Anton A van Zwet ◽  
Arnoud HM van Vliet ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Cell Viability ◽  
Protein Synthesis Inhibitor ◽  
Low Oxygen ◽  
Anaerobic Conditions ◽  
Metronidazole Resistance ◽  
Oxygen Conditions ◽  
H Pylori

Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.

scholarly journals Parameters Affecting Continuous In Vitro Culture of Treponema pallidum Strains

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Diane G. Edmondson ◽  
Bridget D. DeLay ◽  
Lindsay E. Kowis ◽  
Steven J. Norris
Keyword(s):  
Culture System ◽  
Treponema Pallidum ◽  
Basal Medium ◽  
Human Pathogen ◽  
Low Oxygen ◽  
Term Survival ◽  
Content Type ◽  
Long Term Culture

ABSTRACT The bacterium that causes syphilis, Treponema pallidum subsp. pallidum, has now been cultured in vitro continuously for periods exceeding 3 years using a system consisting of coculture with Sf1Ep rabbit epithelial cells in TpCM-2 medium and a low-oxygen environment. In addition, long-term culture of several other syphilis isolates (SS14, Mexico A, UW231B, and UW249B) and the T. pallidum subsp. endemicum Bosnia A strain has been achieved. During in vitro passage, T. pallidum subsp. pallidum exhibited a typical bacterial growth curve with logarithmic and stationary phases. Sf1Ep cells are required for sustained growth and motility; however, high initial Sf1Ep cell numbers resulted in reduced multiplication and survival. Use of Eagle’s minimal essential medium as the basal medium was not effective in sustaining growth of T. pallidum subsp. pallidum beyond the first passage, whereas CMRL 1066 or M199 supported long-term culture, confirming that additional nutrients present in these more complex basal media are required for long-term culture. T. pallidum subsp. pallidum growth was dependent upon the presence of fetal bovine serum, with 20% (vol/vol) being the optimal concentration. Omission of reactive oxygen species scavengers dithiothreitol, d-mannitol, or l-histidine did not dramatically affect survival or growth. Additionally, T. pallidum subsp. pallidum can be successfully cultured in a Brewer jar instead of a specialized low-oxygen incubator. Phosphomycin or amphotericin B can be added to the medium to aid in the prevention of bacterial or fungal contamination, respectively. These results help define the parameters of the T. pallidum subsp. pallidum culture system that are required for sustained, long-term survival and multiplication. IMPORTANCE Syphilis is caused by the bacterium Treponema pallidum subsp. pallidum. Until recently, this pathogen could only be maintained through infection of rabbits or other animals, making study of this important human pathogen challenging and costly. T. pallidum subsp. pallidum has now been successfully cultured for over 3 years in a tissue culture system using a medium called TpCM-2. Here, we further define the growth requirements of this important human pathogen, promoting a better understanding of the biology of this fastidious organism.

scholarly journals Bifidobacterium dentiumFortifies the Intestinal Mucus Layer via Autophagy and Calcium Signaling Pathways

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Melinda A. Engevik ◽  
Berkley Luk ◽  
Alexandra L. Chang-Graham ◽  
Anne Hall ◽  
Beatrice Herrmann ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Signaling Pathways ◽  
Goblet Cell ◽  
Cell Function ◽  
Goblet Cells ◽  
Mucus Layer ◽  
Content Type ◽  
Intestinal Mucus ◽  
Intestinal Mucus Layer

ABSTRACTMuch remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer.Bifidobacteriumspecies colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, selectBifidobacteriumstrains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived speciesBifidobacterium dentiumwould increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans.In silicodata revealed thatB. dentiumlacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating thatB. dentiumcould not use naive mucin glycans as primary carbon sourcesin vitro. To examineB. dentiummucus modulationin vivo, Swiss Webster germfree mice were monoassociated with live or heat-killedB. dentium. LiveB. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markersKrüppel-like factor 4(Klf4),Trefoil factor 3(Tff3),Relm-β,Muc2, and several glycosyltransferases compared to both heat-killedB. dentiumand germfree counterparts. Likewise, liveB. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining.In vitro,B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified thatB. dentium-secreted products, such as γ-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates thatB. dentiumis capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production.IMPORTANCEMicrobe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe,Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike otherBifidobacteriumspecies,B. dentiumdoes not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of usingB. dentiumand similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.

The faecal microbial population can be representative of large intestinal microfloral activity

1998 ◽  
Vol 1998 ◽  
pp. 165-165 ◽  
Author(s):  
B.A. Williams ◽  
C. Voigt ◽  
M.W.A. Verstegen
Keyword(s):  
Large Intestine ◽  
Resistant Starch ◽  
Microbial Population ◽  
Potato Starch ◽  
Gas Production ◽  
Strictly Anaerobic ◽  
Anaerobic Conditions ◽  
Oat Hulls ◽  
Colon And Rectum

Faeces may be representative of the microbial population present in the large intestine (LI) of monogastric animals, and is being used as the inoculum for in vitro procedures to investigate hindgut fermentation. To test this hypothesis, samples were taken from the caecum, mid-colon, and rectum of three pigs fed a simple diet (no antibiotics or copper). The in vitro cumulative gas production technique (Theodorou et a/., 1994) was used to measure the fermentation characteristics of four standard carbohydrate feedstuffs.Four feeds representative of certain carbohydrate fractions were used. They were fructo-oligosaccharide (FOS- oligosaccharides), oat hulls (OatH- fibre), potato starch (PST- resistant starch), and wheat bran (WBR- fibre). The entire large intestine was taken to laboratory, where samples were removed from the caecum, mid-colon, and rectum, diluted, and mixed, under strictly anaerobic conditions before being used as inoculum. The cumulative gas data (144 hours) were fitted to the monophasic modified Michaelis-Menten model (Groot et a/., 1996). After fermentation, samples were taken for VFA analysis, and substrate losses and pH measured.

scholarly journals A SimpleIn VitroGut Model for Studying the Interaction betweenEscherichia coliand the Intestinal Commensal Microbiota in Cecal Mucus

2018 ◽  
Vol 84 (24) ◽  
Author(s):  
Matthew E. Mokszycki ◽  
Mary Leatham-Jensen ◽  
Jon L. Steffensen ◽  
Ying Zhang ◽  
Karen A. Krogfelt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Profiling ◽  
Rrna Gene ◽  
Mucus Layer ◽  
Content Type ◽  
E Coli ◽  
Commensal Microbiota ◽  
Intestinal Mucus

ABSTRACTA novelin vitrogut model was developed to better understand the interactions betweenEscherichia coliand the mouse cecal mucus commensal microbiota. The gut model is simple and inexpensive while providing an environment that largely replicates the nonadherent mucus layer of the mouse cecum. 16S rRNA gene profiling of the cecal microbial communities of streptomycin-treated mice colonized withE. coliMG1655 orE. coliNissle 1917 and the gut model confirmed that the gut model properly reflected the community structure of the mouse intestine. Furthermore, the results from thein vitrogut model mimic the results of publishedin vivocompetitive colonization experiments. The gut model is initiated by the colonization of streptomycin-treated mice, and then the community is serially transferred in microcentrifuge tubes in an anaerobic environment generated in anaerobe jars. The nutritional makeup of the cecum is simulated in the gut model by using a medium consisting of porcine mucin, mouse cecal mucus, HEPES-Hanks buffer (pH 7.2), Cleland’s reagent, and agarose. Agarose was found to be essential for maintaining the stability of the microbial community in the gut model. The outcome of competitions betweenE. colistrains in thein vitrogut model is readily explained by the “restaurant hypothesis” of intestinal colonization. This simple model system potentially can be used to more fully understand how different members of the microbiota interact physically and metabolically during the colonization of the intestinal mucus layer.IMPORTANCEBoth commensal and pathogenic strains ofEscherichia coliappear to colonize the mammalian intestine by interacting physically and metabolically with other members of the microbiota in the mucus layer that overlays the cecal and colonic epithelium. However, the use of animal models and the complexity of the mammalian gut make it difficult to isolate experimental variables that might dictate the interactions betweenE. coliand other members of the microbiota, such as those that are critical for successful colonization. Here, we describe a simple and relatively inexpensivein vitrogut model that largely mimicsin vivoconditions and therefore can facilitate the manipulation of experimental variables for studying the interactions ofE. coliwith the intestinal microbiota.

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