Suppression of Intestinal Epithelial Cell Chemokine Production by Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0389 Is Mediated by Secreted Bioactive Molecules

Host intestinal epithelial cells (IEC) present at the gastrointestinal interface are exposed to pathogenic and non-pathogenic bacteria and their products. Certain probiotic lactic acid bacteria (LAB) have been associated with a range of host-immune modulatory activities including down-regulation of pro-inflammatory gene expression and cytokine production by IEC, with growing evidence suggesting that these bacteria secrete bioactive molecules with immunomodulatory activity. The aim of this study was to determine whether two lactobacilli with immunomodulatory activity [Lactobacillus rhamnosus R0011 (Lr) and Lactobacillus helveticus R0389 (Lh)], produce soluble mediators able to influence IEC responses to Pattern Recognition Receptor (PRR) ligands and pro-inflammatory cytokines [Tumor Necrosis Factor α (TNFα), Interleukin-1β (IL-1β)], signals inducing IEC chemokine production during infection. To this end, the effects of cell-free supernatants (CFS) from Lr and Lh on IEC production of the pro-inflammatory chemokines interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant 1 (CINC-1) induced by a range of host- or pathogen-derived pro-inflammatory stimuli were determined, and the impact on human HT-29 IEC and a primary IEC line (rat IEC-6) was compared. The Lr-CFS and Lh-CFS did not significantly modulate basal IL-8 production from HT-29 IECs or CINC-1 production from IEC-6 cells. However, both Lr-CFS and Lh-CFS significantly down-regulated IL-8 production from HT-29 IECs challenged with varied PRR ligands. Lr-CFS and Lh-CFS had differential effects on PRR-induced CINC-1 production by rat IEC-6 IECs, with no significant down-regulation of CINC-1 observed from IEC-6 IECs cultured with Lh-CFS. Further analysis of the Lr-CFS revealed down-regulation of IL-8 production induced by the pro-inflammatory cytokines IL-1β and TNFα Preliminary characterization of the bioactive constituent(s) of the Lr-CFS indicates that it is resistant to treatment with DNase, RNase, and an acidic protease, but is sensitive to alterations in pH. Taken together, these results indicate that these lactobacilli secrete bioactive molecules of low molecular weight that may modulate host innate immune activity through interactions with IEC

Diversity of oxygenase genes from Methane- and Ammonia-oxidizing bacteria in the Eastern Snake River Plain aquifier

PCR amplification, restriction fragment length polymorphism, and phylogenetic analysis of oxygenase genes were used for the characterization of in situ methane- and ammonia-oxidizing bacteria from free-living and attached communities in the Eastern Snake River Plain aquifer. The following three methane monooxygenase (MMO) PCR primer sets were used: A189-A682, which amplifies an internal region of both the pmoA gene of the MMO particulate form and the amoA gene of ammonia monooxygenase; A189-mb661, which specifically targets the pmoA gene; and mmoXA-mmoXB, which amplifies the mmoX gene of the MMO soluble form (sMMO). Whole-genome amplification (WGA) was used to amplify metagenomic DNA from each community to assess its applicability for generating unbiased metagenomic template DNA. The majority of sequences in each archive were related to oxygenases of type II-like methanotrophs of the genus Methylocystis. A small subset of type I sequences found only in free-living communities possessed oxygenase genes that grouped nearest to Methylobacter and Methylomonas spp. Sequences similar to that of the amoA gene associated with ammoniaoxidizing bacteria (AOB) most closely matched a sequence from the uncultured bacterium BS870 but showed no substantial alignment to known cultured AOB. Based on these functional gene analyses, bacteria related to the type II methanotroph Methylocystis sp. were found to dominate both free-living and attached communities. Metagenomic DNA amplified by WGA showed characteristics similar to those of unamplified samples. Overall, numerous sMMO-like gene sequences that have been previously associated with high rates of trichloroethylene cometabolism were observed in both free-living and attached communities in this basaltic aquifer.Copyrighted by American Society for Microbiology

Isolation and characterization of potent antifungal strains of the Streptomyces violaceusniger clade active against Candida albicans

Streptomyces strains were isolated from a sagebrush rhizosphere soil sample on humic acid vitamin (HV) agar and water yeast extract (WYE) agar supplemented with 1.5% (w/w) phenol as a selective medium. Acidic, neutral and alkaline pH conditions were also used in the isolation procedures. The phenol treatment reduced the numbers of both actinomycetes and non-actinomycetes on plates under all three pH conditions. From phenol-amended HV and WYE agar, 16 strains were isolated in pure culture; 14 from the HV agar and two from the WYE agar. All the isolates were tested for their antifungal activities against Pythium ultimum P8 and five yeast strains, including two antifungal drug-resistant Candida albicans strains. HV isolates that showed broad-spectrum antifungal antibiotic activities were all found to be members of the Streptomyces violaceusniger clade, while those that did not were non-clade members. The phenol treatment was not selective for S. violaceusniger clade members. Therefore, we tested the spores of both S. violaceusniger clade and non-clade members using two biocides, phenol and hydrogen peroxide, as selection agents. Spores of non-clade members, such as S. coelicolor M145 and S. lividans TK 21, survived these two biocides just as well as S. violaceusniger clade members. Thus, in our hands, biocide resistance was not S. violaceusniger clade specific as previously reported. However, isolates showing broad-spectrum antifungal and antiyeast activity were all members of the clade. We conclude that screening of isolates for broad-spectrum antifungal/antiyeast activity is the preferred method of isolating S. violaceusniger clade strains rather than biocide-based selection. Phylogenetic analysis of the phenol-resistant isolates revealed that the HV isolates that exhibited broad-spectrum antifungal antibiotic activity were all clustered and closely related to the S. violaceusniger clade, while the isolates that did not exhibit antifungal antibiotic activity were all non-clade members

Characterization of pellicle inhibition in Gluconacetobacter xylinus 53582 by a small molecule, pellicin, identified by a chemical genetics screen.

Pellicin ([2E]-3-phenyl-1-[2,3,4,5-tetrahydro-1,6-benzodioxocin-8-yl]prop-2-en-1-one) was identified in a chemical genetics screen of 10,000 small molecules for its ability to completely abolish pellicle production in Gluconacetobacter xylinus. Cells grown in the presence of pellicin grew 1.5 times faster than untreated cells. Interestingly, growth in pellicin also caused G. xylinus cells to elongate. Measurement of cellulose synthesis in vitro showed that cellulose synthase activity was not directly inhibited by pellicin. Rather, when cellulose synthase activity was measured in cells that were pre-treated with the compound, the rate of cellulose synthesis increased eight-fold over that observed for untreated cells. This phenomenon was also apparent in the rapid production of cellulose when cells grown in the presence of pellicin were washed and transferred to media lacking the inhibitor. The rate at which cellulose was produced could not be accounted for by growth of the organism. Pellicin was not detected when intracellular contents were analyzed. Furthermore, it was found that pellicin exerts its effect extracellularly by interfering with the crystallization of pre-cellulosic tactoidal aggregates. This interference of the crystallization process resulted in enhanced production of cellulose II as evidenced by the ratio of acid insoluble to acid soluble product in in vitro assays and confirmed in vivo by scanning electron microscopy and powder X-ray diffraction. The relative crystallinity index, RCI, of pellicle produced by untreated G. xylinus cultures was 70% while pellicin-grown cultures had RCI of 38%. Mercerized pellicle of untreated cells had RCI of 42%, which further confirms the mechanism of action of pellicin as an inhibitor of the cellulose I crystallization process. Pellicin is a useful tool for the study of cellulose biosynthesis in G. xylinus

Pellicin affects the cell length of <i>G. xylinus</i>.

<p>Box plot of cell lengths of <i>G. xylinus</i> grown in the presence and absence of pellicin. The box signifies the upper and lower quartiles for each growth condition and the median is represented by the black line within each box. Outliers are denoted by (○). Extreme outliers are denoted by (*). Cell lengths of 500 cells from each growth condition were measured (p<0.001, independent samples <i>t</i>-test).</p

The influence of pellicin on <i>G. xylinus</i> growth.

<p>Pellicin does not affect the viability of <i>G. xylinus</i>. <i>G. xylinus</i> was grown at 30°C in Schramm-Hestrin broth containing 0.1% cellulase and either 10 µM pellicin (▪) or DMSO (▴) as a control. Data show the mean ± SD of four experimental determinations.</p

<i>In vitro</i> cellulose synthase assay using membrane preparations of <i>G. xylinus</i> grown in the presence and absence of pellicin using UDP-[³H]glucose as substrate.

<p>1) Boiled control 2) Untreated 3) Pellicin pretreated. Cells grown in the presence of pellicin show an increased cellulose synthase activity. Data show the mean ± SE of three experimental determinations.</p

Effect of pellicin on cellulose production in liquid culture and solid medium.

<p><i>G. xylinus was</i> grown at 30°C in liquid culture under agitated (A, B) and static conditions (C, D); (A, C) are DMSO controls, (B, D) grown in the presence of 10 µM pellicin. Arrows in (A, C) indicate either the large aggregates or pellicle that form in the absence of pellicin. Colony morphology of <i>G. xylinus</i> grown on SH agar plates that were supplemented with (E, G) DMSO or (F, H) 10 µM pellicin. Photographs of (E, F) were taken with illumination from above and (G, H) were taken with illumination from below of the same colonies. Note the larger, undulate, raised colonies forming on pellicin supplemented plates (F). Arrows in (G) indicate the filiform projections emerging from colony, which are absent in (H). Scale bars equal 0.5 mm.</p