Development of a luciferase-based reporter system to monitor Bifidobacterium breve UCC2003 persistence in mice

<p>Abstract</p> <p>Background</p> <p>Probiotics such as bifidobacteria have been shown to maintain a healthy intestinal microbial balance and help protect against infections. However, despite these benefits, bifidobacteria still remain poorly understood at the biochemical, physiological and especially the genetic level. Herein we describe, for the first time, the development of a non-invasive luciferase-based reporter system for real-time tracking of <it>Bifidobacterium </it>species <it>in vivo</it>.</p> <p>Results</p> <p>The reporter vector pLuxMC1 is based on the recently described theta-type plasmid pBC1 from <it>B. catenatulatum </it><abbrgrp><abbr bid="B1">1</abbr></abbrgrp> and the <it>luxABCDE </it>operon from pPL2lux <abbrgrp><abbr bid="B2">2</abbr></abbrgrp>. Derivatives of pLuxMC1, harbouring a bifidobacterial promoter (pLuxMC2) as well as a synthetically derived promoter (pLuxMC3) <abbrgrp><abbr bid="B3">3</abbr></abbrgrp> placed upstream of <it>luxABCDE</it>, were constructed and found to stably replicate in <it>B. breve </it>UCC2003. The subsequent analysis of these strains allowed us to assess the functionality of pLuxMC1 both <it>in vitro </it>and <it>in vivo</it>.</p> <p>Conclusion</p> <p>Our results demonstrate the potential of pLuxMC1 as a real-time, non-invasive reporter system for <it>Bifidobacterium</it>. It has also allowed us, for the first time, to track the colonisation potential and persistence of this probiotic species in real time. An interesting and significant outcome of the study is the identification of the caecum as a niche environment for <it>B. breve </it>UCC2003 within the mouse gastrointestinal tract (GI) tract.</p

A genome-wide function of THSC/TREX-2 at active genes prevents transcription–replication collisions

The THSC/TREX-2 complex of Saccharomyces cerevisiae mediates the anchoring of transcribed genes to the nuclear pore, linking transcription elongation with mRNA export and genome stability, as shown for specific reporters. However, it is still unknown whether the function of TREX-2 is global and the reason for its relevant role in genome integrity. Here, by studying two TREX-2 representative subunits, Thp1 and Sac3, we show that TREX-2 has a genome-wide role in gene expression. Both proteins show similar distributions along the genome, with a gradient disposition at active genes that increases towards the 3 end. Thp1 and Sac3 have a relevant impact on the expression of long, G+C-rich and highly transcribed genes. Interestingly, replication impairment detected by the genome-wide accumulation of the replicative Rrm3 helicase is increased preferentially at highly expressed genes in the thp1Δ and sac3Δ mutants analyzed. Therefore, our work provides evidence of a function of TREX-2 at the genome-wide level and suggests a role for TREX-2 in preventing transcription– replication conflicts, as a source of genome instability derived from a defective messenger ribonucleoprotein particle (mRNP) biogenesis.Spanish Ministry of Economy and Competitiveness [BFU2010-16372]; Junta de Andalucía [CVI4567 and P12/BIO-1238]; European Union (FEDER); and a JAE predoctoral training grant from the Spanish Research Council (CSIC) [to J.M.S-P.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2010-16372].Peer reviewe

Interleukin-11 alters placentation and causes preeclampsia features in mice

Preeclampsia is an insidious disease, unique to humans, affecting ∼8% of pregnancies. There are no early detection tests or pharmacological treatments. Impaired placentation is widely accepted to contribute to the pathogenesis. However, the mechanisms remain elusive, given the complications of studying first-trimester placental development in women. A major limitation for the study of new treatments is the lack of available animal models that recapitulate the full spectrum of preeclampsia features. We have developed a mouse model characterized by elevated levels of the cytokine Interleukin-11 (IL11). This study provides evidence of a novel pathway causative of preeclampsia features in vivo. It also provides a novel in vivo mouse model that is useful for preclinical studies to test potential therapeutics

High Resolution In Vivo Bioluminescent Imaging for the Study of Bacterial Tumour Targeting

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation

The role of sex hormones in adiposity and insulin resistance in the Aromatase Knockout mouse (ArKO)model

Sex hormones such as estrogens and androgens are known to influence the development of adiposity and insulin resistance, which contribute to many disease processes including the Metabolic Syndrome. Adipose tissue plays a crucial role in energy homeostasis. It was once thought as merely an organ for lipid storage, but it is now regarded as an endocrine organ (Kershaw and Flier, 2004). Adiposity seen in obesity, is linked to over-nutrition and decreased energy output, which leads to an increase in the size and number of adipocytes. This results in the release of adipokines and excess free fatty acids into the circulation and subsequent ectopic accumulation of excess lipids in peripheral tissue eg. skeletal muscle and liver. This can influence insulin sensitivity by causing inflammation and interrupting the insulin signaling pathways leading to decreased glucose uptake (Nawrocki and Scherer, 2004). To research the effects of sex hormones on adiposity and insulin resistance, we studied Aromatase knockout (ArKO) mice which are unable to convert androgens to estrogens. In this thesis we defined the individual and combined roles of estrogens, androgens, progesterone and HRT drug Tibolone in the modulation of adipose tissue accumulation and lipid production in the absence of aromatase. We discovered the estrogenic component of Tibolone was the significant factor in reducing adiposity. Our study has confirmed the efficacy of tibolone as an HRT to reduce adipose tissue accumulation following menopause and also shows that aromatisation of tibolone is not required to elicit these estrogenic effects. Our data confirmed a positive correlation between estrogen, adipose tissue loss and consequently the regulation of leptin in ArKO mice. Sexually dimorphic differences were observed in the levels of adiponectin, TNFα and IL6. We detected an unexpected increase in levels of pro-inflammatory adipokines and decreases in adiponectin levels after estrogen treatment only in the female ArKO mice, despite reduced adiposity. This study therefore presents vital information as to the sexually dimorphic role of estrogen on adipokine regulation. We also discovered sexually dimorphic differences in the ArKO insulin resistance phenotype. Male ArKO mice developed and maintained glucose and pyruvate intolerance and insulin resistance at all ages. This was attributed to the development of hepatic insulin resistance, affecting gluconeogenesis and insulin signaling. Estrogen replacement was able to improve all aspects of this phenotype. Female ArKO mice developed and maintained only glucose intolerance, without developing pyruvate intolerance or insulin resistance, throughout a twelve-month life span. Contrary to males, estrogen replacement had an unexpected effect on the three month-old female ArKO mice, causing insulin resistance and further glucose intolerance. However, by six and twelve months of age, estrogen was able to improve all phenotypes. Lastly, we also noted novel decreases in two microRNAs the omental adipose tissue of the ArKO mice, which target genes involved in lipid metabolism and insulin signalling. These changes suggest a role for estrogen in post-transcriptional modification. Understanding the mechanisms by which sex hormones act on the metabolic pathways will provide insights into understating the pathogenesis of metabolic syndrome established in sex hormone related conditions such as menopaus

The role of sex hormones in adiposity and insulin resistance in the Aromatase Knockout mouse (ArKO)model

Sex hormones such as estrogens and androgens are known to influence the development of adiposity and insulin resistance, which contribute to many disease processes including the Metabolic Syndrome. Adipose tissue plays a crucial role in energy homeostasis. It was once thought as merely an organ for lipid storage, but it is now regarded as an endocrine organ (Kershaw and Flier, 2004). Adiposity seen in obesity, is linked to over-nutrition and decreased energy output, which leads to an increase in the size and number of adipocytes. This results in the release of adipokines and excess free fatty acids into the circulation and subsequent ectopic accumulation of excess lipids in peripheral tissue eg. skeletal muscle and liver. This can influence insulin sensitivity by causing inflammation and interrupting the insulin signaling pathways leading to decreased glucose uptake (Nawrocki and Scherer, 2004). To research the effects of sex hormones on adiposity and insulin resistance, we studied Aromatase knockout (ArKO) mice which are unable to convert androgens to estrogens. In this thesis we defined the individual and combined roles of estrogens, androgens, progesterone and HRT drug Tibolone in the modulation of adipose tissue accumulation and lipid production in the absence of aromatase. We discovered the estrogenic component of Tibolone was the significant factor in reducing adiposity. Our study has confirmed the efficacy of tibolone as an HRT to reduce adipose tissue accumulation following menopause and also shows that aromatisation of tibolone is not required to elicit these estrogenic effects. Our data confirmed a positive correlation between estrogen, adipose tissue loss and consequently the regulation of leptin in ArKO mice. Sexually dimorphic differences were observed in the levels of adiponectin, TNFα and IL6. We detected an unexpected increase in levels of pro-inflammatory adipokines and decreases in adiponectin levels after estrogen treatment only in the female ArKO mice, despite reduced adiposity. This study therefore presents vital information as to the sexually dimorphic role of estrogen on adipokine regulation. We also discovered sexually dimorphic differences in the ArKO insulin resistance phenotype. Male ArKO mice developed and maintained glucose and pyruvate intolerance and insulin resistance at all ages. This was attributed to the development of hepatic insulin resistance, affecting gluconeogenesis and insulin signaling. Estrogen replacement was able to improve all aspects of this phenotype. Female ArKO mice developed and maintained only glucose intolerance, without developing pyruvate intolerance or insulin resistance, throughout a twelve-month life span. Contrary to males, estrogen replacement had an unexpected effect on the three month-old female ArKO mice, causing insulin resistance and further glucose intolerance. However, by six and twelve months of age, estrogen was able to improve all phenotypes. Lastly, we also noted novel decreases in two microRNAs the omental adipose tissue of the ArKO mice, which target genes involved in lipid metabolism and insulin signalling. These changes suggest a role for estrogen in post-transcriptional modification. Understanding the mechanisms by which sex hormones act on the metabolic pathways will provide insights into understating the pathogenesis of metabolic syndrome established in sex hormone related conditions such as menopaus

Molecular Dissection of a Bifidobacterial Replicon▿ †

The 2.1-kb cryptic plasmid pCIBAO89 from Bifidobacterium asteroides harbors a 1.4-kb segment which is sufficient for its autonomous replication. The segment is divided into two parts, the presumed replication origin, ori89, and the rep gene encoding the putative 41-kDa Rep89 replication initiation protein. This minimal replication region of pCIBAO89 was functionally dissected by transcriptional analyses as well as by DNA-binding studies, and the information obtained was exploited to create a number of Escherichia coli-Bifidobacterium shuttle vectors capable of transforming various bifidobacteria with an efficiency of up to 106 transformants/μg DNA

Cross-feeding by bifidobacterium breve ucc2003 during co-cultivation with bifidobacterium bifidum prl2010 in a mucin-based medium

Background: Bifidobacteria constitute a specific group of commensal bacteria that commonly inhabit the mammalian gastrointestinal tract. Bifidobacterium breve UCC2003 was previously shown to utilize a variety of plant/diet/host-derived carbohydrates, including cellodextrin, starch and galactan, as well as the mucin and HMO-derived monosaccharide, sialic acid. In the current study, we investigated the ability of this strain to utilize parts of a host-derived source of carbohydrate, namely the mucin glycoprotein, when grown in co-culture with the mucin-degrading Bifidobacterium bifidum PRL2010. Results: B. breve UCC2003 was shown to exhibit growth properties in a mucin-based medium, but only when grown in the presence of B. bifidum PRL2010, which is known to metabolize mucin. A combination of HPAEC-PAD and transcriptome analyses identified some of the possible monosaccharides and oligosaccharides which support this enhanced co-cultivation growth/viability phenotype. Conclusion: This study describes the potential existence of a gut commensal relationship between two bifidobacterial species. We demonstrate the in vitro ability of B. breve UCC2003 to cross-feed on sugars released by the mucin-degrading activity of B. bifidum PRL2010, thus advancing our knowledge on the metabolic adaptability which allows the former strain to colonize the (infant) gut by its extensive metabolic abilities to (co-)utilize available carbohydrate sources