Functional Characterization of Nucleoid Associated Proteins Acting as Global Transcription Factors in Mycobacterium tuberculosis

The fatal lung disease tuberculosis is caused by the airborne Mycobacterium tuberculosis, a versatile pathogen adapted to rapidly changing environments. Instead of being eradicated by phagocytic cells of its human host, bacilli tune macrophages to support their own growth and even mask their presence from the immune system for several decades. Rapid adjustment of gene expression is critical for bacterial survival and heavily relies on nucleoid-associated proteins (NAPs). NAPs contribute to active DNA management by altering the chromosomal topology through bending, bridging and looping the DNA. These conformational changes can bring distant genetic loci into close spatial proximity or influence DNA supercoiling and therefore accessibility of the transcription machinery. Apart from their architectural role, NAPs moreover act as global transcription factors by direct regulation of numerous genes. In M. tuberculosis, five proteins were assigned a role as NAP. Among these, EspR, HupB and Lsr2 are crucial not only for virulence but also for cellular metabolism. This thesis focuses on the NAPs mIHF and H-NS with the objective of determining their function and target regulon. Both proteins were investigated by means of genetic manipulation, phenotype assessment and structural studies, and the efficiency of a potential new anti-tuberculosis drug acting on Lsr2 was assessed. Chrysomycin, described as specific inhibitor of Lsr2-DNA complex formation, was found to intercalate into the DNA. The resulting toxic effect on both its target M. tuberculosis as well as on eukaryotic cells rendered further development of the compound as an anti-tuberculosis drug futile. We demonstrated that Rv3852, formerly annotated as H-NS, does not act as a NAP. Deletion of the rv3852 gene had no effect on the in vitro phenotype of M. tuberculosis, did not alter nucleoid spread nor position and had no influence on virulence in mice. The mIHF protein on the other hand is not only essential for active bacterial growth, but also indispensable for survival. Generation of a conditional knockdown mutant showed that depletion of mIHF led to elongated cells devoid of septa with abnormal DNA localization and finally to cell death. The target regulon of mIHF was thoroughly studied by mapping its binding sites on the bacterial genome and by identifying genes that were differentially expressed upon depletion of the protein. We found that mIHF has a strong effect on virulence gene expression and, similar to EspR, possesses a major binding site upstream of one of the main virulence factor operons espACD. Analysis of the transcriptional response revealed that mIHF is further involved in the bacterial response to the hostâs immune system, including control of nutrient pathways as well as global protein and nucleic acid synthesis. To define how mIHF interacts with DNA and influences its 3D organisation, the protein structure of mIHF was determined by nuclear magnetic resonance spectroscopy. Binding of mIHF introduced left-hand loops into linear as well as supercoiled DNA substrates, therefore unwinding condensed DNA. We identified two DNA binding domains in mIHF and showed that its stability increased substantially upon DNA binding. All together, the findings of this thesis contribute to a better understanding of the complex gene regulatory network of M. tuberculosis, advancing the knowledge necessary to eventually defeat tuberculosis, a disease that has plagued humanity for millennia

Ecology and distribution of Thaumarchaea in the deep hypolimnion of Lake Maggiore

Ammonia-oxidizing Archaea (AOA) play an important role in the oxidation of ammonia in terrestrial, marine, and geothermal habitats, as confirmed by a number of studies specifically focused on those environments. Much less is known about the ecological role of AOA in freshwaters. In order to reach a high resolution at the Thaumarchaea community level, the probe MGI-535 was specifically designed for this study and applied to fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) analysis. We then applied it to a fine analysis of diversity and relative abundance of AOA in the deepest layers of the oligotrophic Lake Maggiore, confirming previous published results of AOA presence, but showing differences in abundance and distribution within the water column without significant seasonal trends with respect to Bacteria. Furthermore, phylogenetic analysis of AOA clone libraries from deep lake water and from a lake tributary, River Maggia, suggested the riverine origin of AOA of the deep hypolimnion of the lake

Rare Variants in BNC2 Are Implicated in Autosomal-Dominant Congenital Lower Urinary-Tract Obstruction

Contains fulltext : 203603.pdf (publisher's version ) (Open Access

Biomarkers of hypothalamic-pituitary-adrenal axis activity in mice lacking 11β-HSD1 and H6PDH

Glucocorticoid concentrations are a balance between production under the negative feedback control and diurnal rhythm of the hypothalamic-pituitary-adrenal (HPA) axis and peripheral metabolism, for example by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyses the reduction of inactive cortisone (11-dehydrocorticosterone (11-DHC) in mice) to cortisol (corticosterone in mice). Reductase activity is conferred upon 11β-HSD1 by hexose-6-phosphate dehydrogenase (H6PDH). 11β-HSD1 is implicated in the development of obesity, and selective 11β-HSD1 inhibitors are currently under development. We sought to address the concern regarding potential up-regulation of the HPA axis associated with inhibition of 11β-HSD1. We assessed biomarkers for allele combinations of 11β-HSD1 and H6PD Hderived fromdouble heterozygous mouse crosses. H6PDH knock out (KO) adrenals were 69% larger than WT while 11β-HSD1 KO and double KO (DKO) adrenals were ~30% larger than WT - indicative of increased HPA axis drive in KO animals. ACTH-stimulated circulating corticosterone concentrations were 2.2-fold higher in H6PDH KO animals and ~1.5-fold higher in 11β-HSD1 KO and DKO animals compared with WT, proportional to the observed adrenal hypertrophy. KOof H6PDH resulted in a substantial increase in urinary DHC metabolites in males (65%) and females (61%). KO of 11β-HSD1 alone or in combination with H6PDH led to significant increases (36 and 42% respectively) in urinary DHC metabolites in females only. Intermediate 11β-HSD1/H6PDH heterozygotes maintained a normal HPA axis. Urinary steroid metabolite profile by gas chromatography/ mass spectrometry as a biomarker assay may be beneficial in assaying HPA axis status clinically in cases of congenital and acquired 11β-HSD1/H6PDH deficiency

11β-Hydroxysteroid dehydrogenase type 1 contributes to the balance between 7-keto- and 7-hydroxy-oxysterols in vivo

Abstract11β-Hydroxysteroid dehydrogenase 1 (11βHSD1; EC 1.1.1.146) generates active glucocorticoids from inert 11-keto metabolites. However, it can also metabolize alternative substrates, including 7β-hydroxy- and 7-keto-cholesterol (7βOHC, 7KC). This has been demonstrated in vitro but its consequences in vivo are uncertain. We used genetically modified mice to investigate the contribution of 11βHSD1 to the balance of circulating levels of 7KC and 7βOHC in vivo, and dissected in vitro the kinetics of the interactions between oxysterols and glucocorticoids for metabolism by the mouse enzyme.Circulating levels of 7KC and 7βOHC in mice were 91.3±22.3 and 22.6±5.7nM respectively, increasing to 1240±220 and 406±39nM in ApoE−/− mice receiving atherogenic western diet. Disruption of 11βHSD1 in mice increased (p<0.05) the 7KC/7βOHC ratio in plasma (by 20%) and also in isolated microsomes (2 fold). The 7KC/7βOHC ratio was similarly increased when NADPH generation was restricted by disruption of hexose-6-phosphate dehydrogenase.Reduction and oxidation of 7-oxysterols by murine 11βHSD1 proceeded more slowly and substrate affinity was lower than for glucocorticoids. in vitro 7βOHC was a competitive inhibitor of oxidation of corticosterone (Ki=0.9μM), whereas 7KC only weakly inhibited reduction of 11-dehydrocorticosterone. However, supplementation of 7-oxysterols in cultured cells, secondary to cholesterol loading, preferentially slowed reduction of glucocorticoids, rather than oxidation.Thus, in mouse, 11βHSD1 influenced the abundance and balance of circulating and tissue levels of 7βOHC and 7KC, promoting reduction of 7KC. In health, 7-oxysterols are unlikely to regulate glucocorticoid metabolism. However, in hyperlipidaemia, 7-oxysterols may inhibit glucocorticoid metabolism and modulate signaling through corticosteroid receptors

Animal Welfare Payments and Veterinary and Insemination Costs for Dairy Cows

To promote the provision of animal-friendly housing and management exceeding the minimal legal standards, the Swiss government offers direct payments through two programs for several farm animal species. In dairy cows the BTS program pays for group housing systems with a comfortable lying area separated from the feeding area. The other program, the RAUS, requires that cows receive regular exercise in an outdoor run in the winter and a pasture during summer. The aim of the study was to analyze the relationship between the two Swiss direct payment programs and the veterinary and insemination costs for dairy cows. We used a large sample of more than 21,000 dairy farm observations from 2004 to 2014 obtained from the Swiss Farm Accountancy Data Network. A propensity score weighting was combined with a linear regression model to estimate the doubly robust treatment effects of the BTS and/or RAUS programs on dairying and breeding. Compared to the control group, that is, farms participating in neither program, farms in the RAUS tended to reduce their veterinary costs by 2% (CHF 4.71). Participation in both the BTS and RAUS programs resulted in a 10% cost reduction (CHF 19.32). An analysis of the effects of participation in both programs, with farms participating in only the RAUS as the control group, indicated a cost reduction of 7% for the farms participating in both programs (CHF 13.54). In contrast, participation in the RAUS only or in the RAUS and the BTS did not have a significant effect on insemination costs. The results thus indicate that the implementation of higher welfare standards can have a positive effect on the economic situation of a farm

Essential Nucleoid Associated Protein mIHF (Rv1388) Controls Virulence and Housekeeping Genes in Mycobacterium tuberculosis

Tight control of gene expression is crucial for Mycobacterium tuberculosis to adapt to the changing environments encountered when infecting or exiting human cells. While three nucleoid associated proteins (NAPs) EspR, HupB and Lsr2 have been investigated, the role of a fourth, the mycobacterial integration host factor (mIHF), remains elusive. Here, we report a multidisciplinary functional analysis that exploits a conditional mIHF mutant. Gene silencing was bactericidal and resulted in elongated cells devoid of septa, with only one nucleoid. ChIP-sequencing identified 153 broad peaks distributed around the chromosome, which were often situated upstream of transcriptional start sites where EspR also bound. RNA-sequencing showed expression of 209 genes to be heavily affected upon mIHF depletion, including those for many tRNAs, DNA synthesis and virulence pathways. Consistent with NAP function, mIHF acts as a global regulator by directly and indirectly controlling genes required for pathogenesis and for housekeeping functions

EspL is essential for virulence and stabilizes EspE, EspF and EspH levels in Mycobacterium tuberculosis.

The ESX-1, type VII, secretion system represents the major virulence determinant of Mycobacterium tuberculosis, one of the most successful intracellular pathogens. Here, by combining genetic and high-throughput approaches, we show that EspL, a protein of 115 amino acids, is essential for mediating ESX-1-dependent virulence and for stabilization of EspE, EspF and EspH protein levels. Indeed, an espL knock-out mutant was unable to replicate intracellularly, secrete ESX-1 substrates or stimulate innate cytokine production. Moreover, proteomic studies detected greatly reduced amounts of EspE, EspF and EspH in the espL mutant as compared to the wild type strain, suggesting a role for EspL as a chaperone. The latter conclusion was further supported by discovering that EspL interacts with EspD, which was previously demonstrated to stabilize the ESX-1 substrates and effector proteins, EspA and EspC. Loss of EspL also leads to downregulation in M. tuberculosis of WhiB6, a redox-sensitive transcriptional activator of ESX-1 genes. Overall, our data highlight the importance of a so-far overlooked, though conserved, component of the ESX-1 secretion system and begin to delineate the role played by EspE, EspF and EspH in virulence and host-pathogen interaction

Structural and DNA binding properties of mycobacterial integration host factor mIHF

In bacteria, nucleoid associated proteins (NAPs) take part in active chromosome organization by supercoil management, three-dimensional DNA looping and direct transcriptional control. Mycobacterial integration host factor (mIHF, rv1388) is a NAP restricted to Actinobacteria and essential for survival of the human pathogen Mycobacterium tuberculosis. We show in vitro that DNA binding by mIHF strongly stabilizes the protein and increases its melting temperature. The structure obtained by Nuclear Magnetic Resonance (NMR) spectroscopy characterizes mIHF as a globular protein with a protruding alpha helix and a disordered N-terminus, similar to Streptomyces coelicolor IHF (sIHF). NMR revealed no residues of high flexibility, suggesting that mIHF is a rigid protein overall that does not undergo structural rearrangements. We show that mIHF only binds to double stranded DNA in solution, through two DNA binding sites (DBSs) similar to those identified in the X-ray structure of sIHF. According to Atomic Force Microscopy, mIHF is able to introduce left-handed loops of ca. 100 nm size (similar to 300 bp) in supercoiled cosmids, thereby unwinding and relaxing the DNA

Rare Variants in BNC2 Are Implicated in Autosomal-Dominant Congenital Lower Urinary-Tract Obstruction

Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853*]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Resequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage