Toby D. Allen

Enterococcus faecalis Induces Aneuploidy and Tetraploidy in Colonic Epithelial Cells through a Bystander Effect

Intestinal commensals are potential important contributors to the etiology of sporadic colorectal cancer, but mechanisms by which bacteria can initiate tumors remain uncertain. Herein, we describe mechanisms that link Enterococcus faecalis, a bacterium known to produce extracellular superoxide, to the acute induction of chromosomal instability. Immortalized human and nontransformed murine colonic epithelial cells, along with a mouse colonic ligation model, were used to assess the effect of E. faecalis on genomic DNA stability and damage. We found that this human intestinal commensal generated aneuploidy, tetraploidy, and gammaH2AX foci in HCT116, RKO, and YAMC cells. In addition, direct exposure of E. faecalis to these cells induced a G2 cell cycle arrest. Similar observations were noted by exposing cells to E. faecalis-infected macrophages in a dual-chamber coculture system for detecting bystander effects. Manganese superoxide dismutase, catalase, and tocopherols attenuated, and caffeine and inhibitors of glutathione synthase exacerbated, the aneugenic effects and linked the redox-active phenotype of this intestinal commensal to potentially transforming events. These findings provide novel insights into mechanisms by which E. faecalis and intestinal commensals can contribute to cellular transformation and tumorigenesis.

Alkalibaculum bacchi gen. nov., sp. nov., a CO-oxidizing, ethanol-producing acetogen isolated from livestock-impacted soil

Phenotypic and phylogenetic studies were performed on three strains of an acetogenic bacterium isolated from livestock-impacted soil. The bacterium stained Gram-negative and was a non-spore-forming rod that was motile by peritrichous flagella. The novel strains had an optimum pH for growth of 8.0-8.5 and utilized H₂ : CO₂, CO : CO₂, glucose, fructose, mannose, turanose, ribose, trimethylamine, pyruvate, methanol, ethanol, n-propanol and n-butanol as growth substrates. Acetate was produced from glucose. Acetate, CO₂ and ethanol were produced from CO : CO₂. 16S rRNA gene sequence analysis indicated that the novel strains formed a new subline in the family Eubacteriaceae (rRNA cluster XV) of the low G+C-containing Gram-positive bacteria of the class Clostridia. The DNA G+C base composition was 34 mol%. Cell wall analysis revealed the existence of a novel B-type peptidoglycan similar to the B2α-type (B4) configuration with a variation containing aspartic acid. Based on phylogenetic and phenotypic evidence, it is proposed that the new isolates represent a novel genus and species, for which the name Alkalibaculum bacchi gen. nov., sp. nov. is proposed. The type strain of the type species is CP11(T) (=ATCC BAA-1772(T)=DSM 22112(T)).

Desulfovibrio carbinoliphilus sp. nov., a benzyl alcohol-oxidizing, sulfate-reducing bacterium isolated from a gas condensate-contaminated aquifer

Phenotypic and phylogenetic studies were performed on a novel sulfate-reducing bacterium, strain D41(T), isolated as part of a methanogenic syntrophic culture from a gas condensate-contaminated aquifer undergoing intrinsic bioremediation. The bacterium was a Gram-negative, non-spore-forming, curved rod, motile by a single polar flagellum, which oxidized several alcohols incompletely, including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 3-methyl-1-butanol (isoamyl alcohol), ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, phenylethanol and benzyl alcohol. Additionally, the strain oxidized H(2)/CO(2), formate, lactate, pyruvate, maleate, malate and fumarate. Sulfate, thiosulfate and sulfite were used as electron acceptors. The DNA G+C content was 63 mol%. Based on phylogenetic and phenotypic evidence, the novel species Desulfovibrio carbinoliphilus sp. nov. is proposed. The type strain is D41(T) (=ATCC BAA-1241(T) =DSM 17524(T)).

Cyclooxygenase-2 generates the endogenous mutagen trans-4-hydroxy-2-nonenal in Enterococcus faecalis-infected macrophages

Infection of macrophages by the human intestinal commensal Enterococcus faecalis generates DNA damage and chromosomal instability in mammalian cells through bystander effects. These effects are characterized by clastogenesis and damage to mitotic spindles in target cells and are mediated, in part, by trans-4-hydroxy-2-nonenal (4-HNE). In this study, we investigated the role of COX and lipoxygenase (LOX) in producing this reactive aldehyde using E. faecalis–infected macrophages and interleukin (IL)-10–knockout mice colonized with this commensal. 4-HNE production by E. faecalis–infected macrophages was significantly reduced by COX and LOX inhibitors. The infection of macrophages led to decreased Cox1 and Alox5 expression whereas COX-2 and 4-HNE increased. Silencing Alox5 and Cox1 with gene-specific siRNAs had no effect on 4-HNE production. In contrast, silencing Cox2 significantly decreased 4-HNE production by E. faecalis–infected macrophages. Depleting intracellular glutathione increased 4-HNE production by these cells. Next, to confirm COX-2 as a source for 4-HNE, we assayed the products generated by recombinant human COX-2 and found 4-HNE in a concentration-dependent manner using arachidonic acid as a substrate. Finally, tissue macrophages in colon biopsies from IL-10–knockout mice colonized with E. faecalis were positive for COX-2 by immunohistochemical staining. This was associated with increased staining for 4-HNE protein adducts in surrounding stroma. These data show that E. faecalis, a human intestinal commensal, can trigger macrophages to produce 4-HNE through COX-2. Importantly, it reinforces the concept of COX-2 as a procarcinogenic enzyme capable of damaging DNA in target cells through bystander effects that contribute to colorectal carcinogenesis. Cancer Prev Res; 6(3); 206–16. ©2013 AACR.

Anti-Cancer Activity of Nitrones in the Apc Min/+ Model of Colorectal Cancer

Abstract The nitrones of α-phenyl-tert-butyl nitrone (PBN) and 4-hydroxyl-PBN (4-OH-PBN) that have anti-cancer activity in models of liver cancer and glioblastomas were tested in the ApcMin/+ mouse model. Mice were administered PBN and 4-OH-PBN in drinking water and intestinal tumour size and number assessed after 3–4 months. Throughout the experiment, contrast-enhanced magnetic resonance imaging (MRI) was used to monitor colon tumours. MRI data showed a time-dependent significant increase in total colonic signal intensity in sham-treated mice, but a significant decrease for PBN-treated mice and slight decrease for 4-OHPBN treated mice, probably due to the limited water solubility of 4-OH-PBN. Final pathological and percentage survival data agreed with the MRI data. PBN had little effect on oxaliplatin-mediated killing of HCT116 colon cancer cells and caused only a slight decrease in the amount of active fraction caspase 3 in oxaliplatin-treated cells. PBN has significant anti-cancer activity in this model of intestinal neoplasia.

Dichotomous metabolism of Enterococcus faecalis induced by haematin starvation modulates colonic gene expression

Enterococcus faecalis is an intestinal commensal that cannot synthesize porphyrins and only expresses a functional respiratory chain when provided with exogenous haematin. In the absence of haematin, E. faecalis reverts to fermentative metabolism and produces extracellular superoxide that can damage epithelial-cell DNA. The acute response of the colonic mucosa to haematin-starved E. faecalis was identified by gene array. E. faecalis was inoculated into murine colons using a surgical ligation model that preserved tissue architecture and homeostasis. The mucosa was exposed to haematin-starved E. faecalis and compared with a control consisting of the same strain grown with haematin. At 1 h post-inoculation, 6 mucosal genes were differentially regulated and this increased to 42 genes at 6 h. At 6 h, a highly significant biological interaction network was identified with functions that included nuclear factor-kappaB (NF-kappaB) signalling, apoptosis and cell-cycle regulation. Colon biopsies showed no histological abnormalities by haematoxylin and eosin staining. Immunohistochemical staining, however, detected NF-kappaB activation in tissue macrophages using antibodies to the nuclear localization sequence for p65 and the F4/80 marker for murine macrophages. Similarly, haematin-starved E. faecalis strongly activated NF-kappaB in murine macrophages in vitro. Furthermore, primary and transformed colonic epithelial cells activated the G2/M checkpoint in vitro following exposure to haematin-starved E. faecalis. Modulation of this cell-cycle checkpoint was due to extracellular superoxide produced as a result of the respiratory block in haematin-starved E. faecalis. These results demonstrate that the uniquely dichotomous metabolism of E. faecalis can significantly modulate gene expression in the colonic mucosa for pathways associated with inflammation, apoptosis and cell-cycle regulation.

Tolumonas osonensis sp. nov., isolated from anoxic freshwater sediment, and emended description of the genus Tolumonas

A polyphasic taxonomic study was performed on a strain of an unknown Gram-negative, non-motile, saccharolytic, facultatively anaerobic bacterium, strain OCF 7(T), isolated from anoxic freshwater sediment. The strain grew optimally at 22 °C and pH 7.5, and was able to grow under strictly anaerobic conditions. Major fermentation products from glucose metabolism were formate, acetate, ethanol and lactate. Comparative 16S rRNA gene sequence analysis indicated that strain OCF 7(T) was phylogenetically related to the type strain of Tolumonas auensis (97.2 % similarity) within the family Aeromonadaceae of the Gammaproteobacteria. However, OCF 7(T) did not produce toluene from phenylacetate, phenylalanine, phenoxyacetate, phenylsuccinate or phenylbutyrate in the presence of glucose. Phenol was not produced from tyrosine or phenoxyacetate in the presence of glucose. Dominant fatty acids of this micro-organism included C(16 : 0), C(18 : 1)ω7c and C(16 : 1)ω7c (and/or iso-C(15 : 0) 2-OH). Major polar lipids were phosphatidylglycerol and phosphatidylethanolamine, and the respiratory quinone was menaquinone MK-8. The genomic DNA G+C content of strain OCF 7(T) was 52.1 mol%. Based on phylogenetic and phenotypic evidence, strain OCF 7(T) should be classified as a representative of a novel species of Tolumonas, for which the name Tolumonas osonensis sp. nov. is proposed; the type strain is OCF 7(T) ( = DSM 22975(T) = ATCC BAA-1908(T)). An emended description of the genus Tolumonas is also given.

Youngiibacter fragilis gen. nov., sp. nov., isolated from natural gas production-water and reclassification of Acetivibrio multivorans as Youngiibacter multivorans comb. nov.

A taxonomic study employing a polyphasic approach was performed on a novel anaerobic bacterium isolated from natural gas production-water. The bacterium stained Gram-negative and consisted of non-motile, non-spore-forming, rod-shaped cells. Products of glucose or starch fermentation were ethanol, CO2, formate, acetate and H2. The predominant fatty acids were C16 : 0 ALDE and summed feature 3 comprising C16 : 1ω7c and/or C16 : 1ω6c. The DNA G+C content was 45.5 mol%. 16S rRNA gene sequence analysis demonstrated that the nearest phylogenetic neighbours of the novel strain were Acetivibrio multivorans DSM 6139(T) (98.5 %) and Proteiniclasticum ruminis JCM 14817(T) (95.4 %). The DNA-DNA hybridization value between the novel organism and Acetivibrio multivorans PeC1 DSM 6139(T) was determined to be only 30.2 %, demonstrating the separateness of the two species. Based on phylogenetic, phenotypic and chemotaxonomic evidence that clearly distinguished strain 232.1(T) from Proteiniclasticum ruminis and other close relatives, it is proposed that the novel isolate be classified as representing a novel species of a new genus within the family Clostridiaceae, Youngiibacter fragilis gen. nov., sp. nov. The type strain of the type species is 232.1(T) ( = ATCC BAA-2257(T) = DSM 24749(T)). In addition, Acetivibrio multivorans is proposed to be reclassified as Youngiibacter multivorans comb. nov.