The production and detoxification of a potent cytotoxin, nitric oxide, by pathogenic enteric bacteria

The nitrogen cycle is based on several redox reactions that are mainly accomplished by prokaryotic organisms, some archaea and a few eukaryotes, which use these reactions for assimilatory, dissimilatory or respiratory purposes. One group is the Enterobacteriaceae family of Gammaproteobacteria, which have their natural habitats in soil, marine environments or the intestines of humans and other warm-blooded animals. Some of the genera are pathogenic and usually associated with intestinal infections. Our body possesses several physical and chemical defence mechanisms to prevent pathogenic enteric bacteria from invading the gastrointestinal tract. One response of the innate immune system is to activate macrophages, which produce the potent cytotoxin nitric oxide (NO). However, some pathogens have evolved the ability to detoxify NO to less toxic compounds, such as the neuropharmacological agent and greenhouse gas nitrous oxide (N2O), which enables them to overcome the host's attack. The same mechanisms may be used by bacteria producing NO endogenously as a by-product of anaerobic nitrate respiration. In the present review, we provide a brief introduction into the NO detoxification mechanisms of two members of the Enterobacteriaceae family: Escherichia coli and Salmonella enterica serovar Typhimurium. These are discussed as comparative non-pathogenic and pathogenic model systems in order to investigate the importance of detoxifying NO and producing N2O for the pathogenicity of enteric bacteria

Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant

BACKGROUND: Inositol 1,4,5trisphosphate (IP(3)) and diacylglycerol (DAG) are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia), whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. METHODOLOGY/PRINCIPAL FINDINGS: We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3(mNab)) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3(mNab) alleles are phenotypically normal. However, the presence of one Plcd3(mNab) allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the Plcd3(mNab) mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. CONCLUSIONS/SIGNIFICANCE: The Plcd3(mNab) mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface

The Endoplasmic Reticulum Stress Response in Neuroprogressive Diseases: Emerging Pathophysiological Role and Translational Implications

The endoplasmic reticulum (ER) is the main cellular organelle involved in protein synthesis, assembly and secretion. Accumulating evidence shows that across several neurodegenerative and neuroprogressive diseases, ER stress ensues, which is accompanied by over-activation of the unfolded protein response (UPR). Although the UPR could initially serve adaptive purposes in conditions associated with higher cellular demands and after exposure to a range of pathophysiological insults, over time the UPR may become detrimental, thus contributing to neuroprogression. Herein, we propose that immune-inflammatory, neuro-oxidative, neuro-nitrosative, as well as mitochondrial pathways may reciprocally interact with aberrations in UPR pathways. Furthermore, ER stress may contribute to a deregulation in calcium homoeostasis. The common denominator of these pathways is a decrease in neuronal resilience, synaptic dysfunction and even cell death. This review also discusses how mechanisms related to ER stress could be explored as a source for novel therapeutic targets for neurodegenerative and neuroprogressive diseases. The design of randomised controlled trials testing compounds that target aberrant UPR-related pathways within the emerging framework of precision psychiatry is warranted

Living with stress: A lesson from the enteric pathogen salmonella enterica

The ability to sense and respond to the environment is essential for the survival of all living organisms. Bacterial pathogens such as Salmonella enterica are of particular interest due to their ability to sense and adapt to the diverse range of conditions they encounter, both in vivo and in environmental reservoirs. During this cycling from host to non-host environments, Salmonella encounter a variety of environmental insults ranging from temperature fluctuations, nutrient availability and changes in osmolarity, to the presence of antimicrobial peptides and reactive oxygen/nitrogen species. Such fluctuating conditions impact on various areas of bacterial physiology including virulence, growth and antimicrobial resistance. A key component of the success of any bacterial pathogen is the ability to recognize and mount a suitable response to the discrete chemical and physical stresses elicited by the host. Such responses occur through a coordinated and complex programme of gene expression and protein activity, involving a range of transcriptional regulators, sigma factors and two component regulatory systems. This review briefly outlines the various stresses encountered throughout the Salmonella life cycle and the repertoire of regulatory responses with which Salmonella counters. In particular, how these Gram-negative bacteria are able to alleviate disruption in periplasmic envelope homeostasis through a group of stress responses, known collectively as the Envelope Stress Responses, alongside the mechanisms used to overcome nitrosative stress, will be examined in more detail

Evaluation and quantification of spectral information in tissue by confocal microscopy

A confocal imaging and image processing scheme is introduced to visualize and evaluate the spatial distribution of spectral information in tissue. The image data are recorded using a confocal laser-scanning microscope equipped with a detection unit that provides high spectral resolution. The processing scheme is based on spectral data, is less error-prone than intensity-based visualization and evaluation methods, and provides quantitative information on the composition of the sample. The method is tested and validated in the context of the development of dermal drug delivery systems, introducing a quantitative uptake indicator to compare the performances of different delivery systems is introduced. A drug penetration study was performed in vitro. The results show that the method is able to detect, visualize and measure spectral information in tissue. In the penetration study, uptake efficiencies of different experiment setups could be discriminated and quantitatively described. The developed uptake indicator is a step towards a quantitative assessment and, in a more general view apart from pharmaceutical research, provides valuable information on tissue composition. It can potentially be used for clinical in vitro and in vivo applications

Apoptosis in <i>oltNH</i> hair follicles.

<p>Apoptosis was investigated by performing the TUNEL assay on paraffin sections. Apoptosis is visualised by Cy3 fluorescence (red signal). DAPI was used as a nuclear counterstain (blue signal). Sections were taken from skin biopsies of <i>oltNH</i> mice (A to G) from postnatal day 2 to 25. A wild-type skin section on postnatal day 13 is shown as control (H). Three biopsies from different animals were used for each time point investigated. Arrows mark the hair bulb (B) or the trailing ends (T) of regressing hair follicles. Arrowheads point at TUNEL positive cells. There are numerous TUNEL positive cells in the matrix of <i>oltNH</i> hair follicles beginning on days 6 and 8 (arrow heads in B and C) and the regressing follicles on day 11 to 13. On day 25, the <i>oltNH</i> hair follicle re-enters anagen and shows no TUNEL positive cells (arrow in G). Throughout this period, there were no TUNEL positive cells in the hair bulbs of wild-type mice (not shown), only some cells in the inner root sheath exhibited a TUNEL positive signal (arrowheads in H), while the medulla showed unspecific autofluorescence. E, epidermis. Bar in E  = 50 µm for all images.</p

<i>Crisp1</i> expression in wild-type and <i>oltNH</i> hair follicles.

<p>In situ hybridisation with a gene-specific probe for <i>Crisp1</i> on skin sections of wild-type (A and C) and <i>oltNH</i> (B and D) mice on postnatal days 6 (A and B) and 8 (C and D). The DIG-labelled probe was visualised using alkaline phosphatase-conjugated anti DIG antibody. Images were taken in brightfield mode with a Nuance VX camera and further processed by spectral analysis using the accompanying software. The specific in situ signal spectrum was pseudo-coloured in red and the eumelanin spectral signal in black. Three biopsies from different animals were used for each time point investigated. Bar  = 100 µm. <i>Crisp1</i> expression is detected in the medulla of wild-type hair follicles (arrows in A and C, red signal), but not in comparable sections of <i>oltNH</i> mice (arrows in B and D).</p

Histology of the infundibular region and distorted hair shafts.

<p>Methacrylate (Technovit 7100) sections of representative areas in the dorsal skin on postnatal day 9, HE stain. The phenotype (Wt, <i>olt</i> (i.e. <i>Del(9)olt1Pas</i>), <i>oltSH</i> or <i>oltNH</i>, respectively) and genotype with respect to the <i>Plcd1</i> (<i>Plcd1*</i> “-” refers to the <i>Del(9)olt1Pas</i> mutation) and <i>Plcd3</i> (Plcd3** “-” refers to the <i>Plcd3</i>^mNab allele) gene is indicated for each image. At least 4 biopsies of each genotype have been investigated. In E, Bar  = 25 µm. These is no hair loss and are no distorted hair shafts neither in wild-type mice heterozygous for both mutant alleles (A), nor those heterozygous for the <i>Del(9)olt1Pas</i> mutation and homozygous for the <i>Plcd3</i>^mNab allele (B), nor others being wild-type for <i>Plcd1</i> and homozygous for the <i>Plcd3</i>^mNab mutant allele (C). Arrows indicate normal hair shafts. Arrowheads mark distorted hair shafts in <i>Del(9)olt1Pas</i> (<i>olt</i>), <i>oltSH</i> and <i>oltNH</i> mice. The alterations of the hair shafts appear histologically similar in all three mutant specimens.</p