Messenger Levels Research Articles

Microscopic colitis patients demonstrate a mixed Th17/Tc17 and Th1/Tc1 mucosal cytokine profile

BackgroundMicroscopic colitis (MC) is a chronic inflammatory bowel disorder of unknown aetiology comprising collagenous colitis (CC) and lymphocytic colitis (LC). Data on the local cytokine profile in MC is limited. This study investigated the T helper (Th) cell and cytotoxic T lymphocyte (CTL) mucosal cytokine profile at messenger and protein levels in MC patients. MethodsMucosal biopsies from CC (n=10), LC (n=5), and CC or LC patients in histopathological remission (CC-HR, n=4), (LC-HR, n=6), ulcerative colitis (UC, n=3) and controls (n=10) were analysed by real-time PCR and Luminex for expression/production of IL-1β, -4, -5, -6, -10, -12, -17, -21, -22, -23, IFN-γ, TNF-α, T-bet and RORC2. ResultsMucosal mRNA but not protein levels of IFN-γ and IL-12 were significantly up regulated in CC, LC as well as UC patients compared to controls. Transcription of the Th1 transcription factor T-bet was significantly enhanced in CC but not LC patients. mRNA levels for IL-17A, IL-21, IL-22 and IL-6 were significantly up regulated in CC and LC patients compared to controls, albeit less than in UC patients. Significantly enhanced IL-21 protein levels were noted in both CC and LC patients. IL-6 protein and IL-1β mRNA levels were increased in CC and UC but not LC patients. Increased mucosal mRNA levels of IFN-γ, IL-21 and IL-22 were correlated with higher clinical activity, recorded as the number of bowel movements per day, in MC patients.Although at lower magnitude, IL-23A mRNA was upregulated in CC and LC, whereas TNF-α protein was increased in CC, LC as well as in UC patients.Neither mRNA nor protein levels of IL-4, IL-5 or IL-10 were significantly changed in any of the colitis groups. LC-HR and especially CC-HR patients had normalized mRNA and protein levels of the above cytokines compared to LC and CC patients. No significant differences were found between LC and CC in cytokine expression/production. ConclusionLC and CC patients demonstrate a mixed Th17/Tc17 and Th1/Tc1 mucosal cytokine profile.

Integration of the Second Messenger c-di-GMP into the Chemotactic Signaling Pathway

ABSTRACTElevated intracellular levels of the bacterial second messenger c-di-GMP are known to suppress motility and promote sessility. Bacterial chemotaxis guides motile cells in gradients of attractants and repellents over broad concentration ranges, thus allowing bacteria to quickly adapt to changes in their surroundings. Here, we describe a chemotaxis receptor that enhances, as opposed to suppresses, motility in response to temporary increases in intracellular c-di-GMP. Azospirillum brasilense’s preferred metabolism is adapted to microaerophily, and these motile cells quickly navigate to zones of low oxygen concentration by aerotaxis. We observed that changes in oxygen concentration result in rapid changes in intracellular c-di-GMP levels. The aerotaxis and chemotaxis receptor, Tlp1, binds c-di-GMP via its C-terminal PilZ domain and promotes persistent motility by increasing swimming velocity and decreasing swimming reversal frequency, which helps A. brasilense reach low-oxygen zones. If c-di-GMP levels remain high for extended periods, A. brasilense forms nonmotile clumps or biofilms on abiotic surfaces. These results suggest that association of increased c-di-GMP levels with sessility is correct on a long-term scale, while in the short-term c-di-GMP may actually promote, as opposed to suppress, motility. Our data suggest that sensing c-di-GMP by Tlp1 functions similar to methylation-based adaptation. Numerous chemotaxis receptors contain C-terminal PilZ domains or other sensory domains, suggesting that intracellular c-di-GMP as well as additional stimuli can be used to modulate adaptation of bacterial chemotaxis receptors.

Identification of microRNA targets in tomato fruit development using high-throughput sequencing and degradome analysis

MicroRNAs (miRNAs) play important roles in plant development through regulation of gene expression by mRNA degradation or translational inhibition. Despite the fact that tomato (Solanum lycopersicum) is the model system for studying fleshy fruit development and ripening, only a few experimentally proven miRNA targets are known, and the role of miRNA action in these processes remains largely unknown. Here, by using parallel analysis of RNA ends (PARE) for global identification of miRNA targets and comparing four different stages of tomato fruit development, a total of 119 target genes of miRNAs were identified. Of these, 106 appeared to be new targets. A large part of the identified targets (56) coded for transcription factors. Auxin response factors, as well as two known ripening regulators, COLORLESS NON-RIPENING (CNR) and APETALA2a (SlAP2a), with developmentally regulated degradation patterns were identified. The levels of the intact messenger of both CNR and AP2a are actively modulated during ripening, by miR156/157 and miR172, respectively. Additionally, two TAS3-mRNA loci were identified as targets of miR390. Other targets such as ARGONAUTE 1 (AGO1), shown to be involved in miRNA biogenesis in other plant species, were identified, which suggests a feedback loop regulation of this process. In this study, it is shown that miRNA-guided cleavage of mRNAs is likely to play an important role in tomato fruit development and ripening.

MAPK Phosphatase-2 (MKP-2) Is Induced by hCG and Plays a Role in the Regulation of CYP11A1 Expression in MA-10 Leydig Cells

MAPKs such as ERK1/2 are dephosphorylated, and consequently inactivated, by dual specificity phosphatases (MKPs). In Leydig cells, LH triggers ERK1/2 phosphorylation through the action of protein kinase A. We demonstrate that, in MA-10 Leydig cells, LH receptor activation by human chorionic gonadotropin (hCG) up-regulates MKP-2, a phosphatase that dephosphorylates ERK1/2, among other MAPKs. After 2 hours, hCG and 8-bromo-cAMP (8Br-cAMP) significantly increased MKP-2 mRNA levels (3-fold), which declined to basal levels after 6 hours. MKP-2 protein accumulation exhibited a similar kinetic profile. In cells transiently expressing flag-MKP-2 protein, hCG/8Br-cAMP stimulation promoted the accumulation of the chimera (2.5-fold after 3 h of stimulation). Pharmacologic and biochemical approaches showed that the accumulation of flag-MKP-2 involves a posttranslational modification that increases MKP-2 half-life. MKP-2 down-regulation by a short hairpin RNA (MKP-2 shRNA) raised the levels of phosphorylated ERK1/2 reached by 8Br-cAMP stimulation. This effect was evident after 180 min of stimulation, which suggests that MKP-2 down-regulates the late phase of cAMP-induced ERK1/2 activity. Also, MKP-2 down-regulation by MKP-2 shRNA increased the stimulatory effect of 8Br-cAMP on both promoter activity and messenger levels of CYP11A1, which encodes for the steroidogenic enzyme P450scc and is induced by LH/hCG through protein kinase A and ERK1/2 activities. Our findings demonstrate, for the first time, that LH/hCG tightly regulates MKP-2 expression, which modulates the induction of CYP11A1 by 8Br-cAMP. MKP-2 up-regulation might control ERK1/2 activity in a specific temporal frame to modulate the expression of a finite repertory of ERK-dependent genes.

An ACACB Variant Implicated in Diabetic Nephropathy Associates with Body Mass Index and Gene Expression in Obese Subjects

Acetyl coenzyme A carboxylase B gene (ACACB) single nucleotide polymorphism (SNP) rs2268388 is reproducibly associated with type 2 diabetes (T2DM)-associated nephropathy (DN). ACACB knock-out mice are also protected from obesity. This study assessed relationships between rs2268388, body mass index (BMI) and gene expression in multiple populations, with and without T2DM. Among subjects without T2DM, rs2268388 DN risk allele (T) associated with higher BMI in Pima Indian children (n = 2021; p-additive = 0.029) and African Americans (AAs) (n = 177; p-additive = 0.05), with a trend in European Americans (EAs) (n = 512; p-additive = 0.09), but not Germans (n = 858; p-additive = 0.765). Association with BMI was seen in a meta-analysis including all non-T2DM subjects (n = 3568; p-additive = 0.02). Among subjects with T2DM, rs2268388 was not associated with BMI in Japanese (n = 2912) or EAs (n = 1149); however, the T allele associated with higher BMI in the subset with BMI≥30 kg/m2 (n = 568 EAs; p-additive = 0.049, n = 196 Japanese; p-additive = 0.049). Association with BMI was strengthened in a T2DM meta-analysis that included an additional 756 AAs (p-additive = 0.080) and 48 Hong Kong Chinese (p-additive = 0.81) with BMI≥30 kg/m2 (n = 1575; p-additive = 0.0033). The effect of rs2268388 on gene expression revealed that the T risk allele associated with higher ACACB messenger levels in adipose tissue (41 EAs and 20 AAs with BMI>30 kg/m2; p-additive = 0.018) and ACACB protein levels in the liver tissue (mixed model p-additive = 0.03, in 25 EA bariatric surgery patients with BMI>30 kg/m2 for 75 exams). The T allele also associated with higher hepatic triglyceride levels. These data support a role for ACACB in obesity and potential roles for altered lipid metabolism in susceptibility to DN.

The Cholesterol-Binding Antibiotic Nystatin Induces Expression of Macrophage Inflammatory Protein-1 in Macrophages

Nystatin, a polyene antifungal antibiotic, is a cholesterol sequestering agent. The antifungal agent alters composition of the plasma membrane of eukaryotic cells, whereas its effects on cells are poorly investigated. In the current study, we investigated the question of whether nystatin was able to induce expression of macrophage inflammatory protein-1 (MIP-1). THP-1 cells rarely express MIP-1α and MIP-1β, however, upon exposure to nystatin, significantly elevated expression of MIP-1α and MIP-1β was observed in a dose-dependent fashion at the messenger and protein levels. Cellular factors activated by nystatin as well as involved in nystatin-induced expression of MIP-1 proteins were identified in order to understand the molecular mechanisms of action of the anti-fungal agent. Treatment with nystatin resulted in enhanced phosphorylation of Akt, ERK, p38 MAPK, and JNK. Abrogation or significant attenuation of nystatin-induced expression of MIP-1α and MIP-1β was observed by treatment with Akt inhibitor IV, LY294002, and SP6001250. Inhibition of ERK or p38MAPK using U0126 and SB202190 did not lead to attenuation of MIP-1 expression. In addition, inhibitors of protein kinase C, such as GF109203X and Ro-318220, also attenuated expression of MIP-1. These results indicate that nystatin is able to activate multiple cellular kinases and, among them, Akt and JNK play primary roles in nystatin-induced expression of MIP-1 proteins.

Sympathetic Nervous System

Activation of the sympathetic nervous system after myocardial infarction results in the mobilization of hematopoietic stem cells, causing an inflammatory boost that accelerates atherosclerosis. This may have important implications for future therapeutic interventions targeting inflammatory pathways. The social and economic impacts of atherosclerosis and subsequent macrovascular clinical manifestations like myocardial infarction (MI) and stroke are immense. Complications of atherosclerosis and MI, such as heart failure, and also recurrent cardiovascular events present a major challenge. They seem to occur more frequently after MI than expected by presumed linear progression of atherosclerosis. In a recent article in Nature , Dutta et al1 found strong evidence that, like in a vicious cycle, acute MI itself accelerates atherosclerosis by triggering a burst of acute systemic inflammation initiated by progenitor cell mobilization from the bone marrow niche. The authors present 4 major findings: (1) acute MI (but also stroke) activates the sympathetic nervous system (SNS); (2) this causes the release of upstream progenitor cells from bone marrow niches; (3) these cells are hosted by the spleen leading to amplified extramedullary myelopoiesis; and (4) at the end of this multimodal cascade, there is a strong increase in inflammation in atherosclerotic plaques, leading to increased size and instability. The authors also show that experimental MI in apolipoprotein E–/– mice activates inflammatory pathways. In serial measurements after coronary ligation, concentrations of cathepsin in aortic plaques and the expression of the inflammatory cytokines interleukin (IL)-6, myeloperoxidase, and matrix metalloproteinase-9 were increased. Consecutively, this was associated with higher vulnerability of atherosclerotic plaque, as demonstrated by decreased thickness of the fibrous cap, and enlarged necrotic cores. Moreover, after MI, the number of macrophages and monocytes, especially the Ly-6Chigh monocyte subset, were increased in the aorta of apolipoprotein E–/– mice and exhibited higher levels of messenger …

The Influences of Reserpine and Imipramine on the 5-HT2 Receptor Binding Site and Its Coupled Second Messenger in Rat Cerebral Cortex

An investigation on the molecular mechanism of depression state, less attention was focused on changes at the intracellular messenger level. In this study the effects of reserpine, a monoamine depletor, and imipramine, an antidepressant, on serotonin-2 (5-HT2) receptor binding and its second messenger system of rat cerebral cortex were studied. The level of inositol 4-monophosphate (IP1) accumulation elicited by 100 microM 5-HT via activation of the 5-HT2 receptor on cerebral cortical slices at twelve hours after a single dose of reserpine (2 mg/kg, i.p.) was significantly higher in treated rats, when compared to that of saline-treated rats; this significant level lasted for at least four days. The level of IP1 accumulation in rat cerebral cortical slices elicited by 100 microM serotonin was higher in the group pretreated with reserpine (0.25 mg/kg/day) sub-chronically for seven days than the group pretreated with normal saline. In the receptor binding study, the maximum binding (B(max)) of 5-HT2 receptor binding was increased, when compared to the corresponding controls; whereas, the dissociation equilibrium constant (K(d)) value of the 5-HT2 receptor was found unchanged in the reserpine treated group. Increases in the sensitivity of phosphoinositol (PI) turnover coupled with the 5-HT2 receptor were also found in the long-term (21 days) low dose (0.1 mg/kg/day) administration of reserpine. However, a long-term administration of imipramine (10 mg/kg/day) reduced the function of the PI turnover coupled with the 5-HT2 receptor. Results obtained from the combined use of reserpine and imipramine demonstrated that this combination was able to antagonize the super-sensitivity of the second messenger responses in 5-HT2 receptor induced by long-term treatment with reserpine. Long-term treatment with reserpine but not imipramine also caused an increase in the B(max) of the 5-HT2 receptor. This up-regulation of the 5-HT2 receptor by reserpine could be antagonized by imipramine, if a combined treatment was employed. However, this combination of imipramine with an additional phospholipid liposome did not enhance or decrease the imipramine's effect on the 5-HT2 receptor, or on its coupled second messenger level. In summary, reserpine induced up-regulation of the postsynaptic monoamine receptor and its coupled second messenger responses (such as IP1 formation). Imipramine was capable of antagonizing these same events in a depression animal model with reserpine. This study demonstrated the dynamic changes and adaptability of the receptor system, followed by changes in PI turnover. The results provide an explanation at the molecular level for the bases of depression and the role of antidepressant drugs effects on those pathological linking elements.

In vitro studies on the effect of 8-(4-chlorophenylthio) cAMP on selected biochemical parameters in Sertoli cells isolated from testis of transgenic mice

The central objective of present study was to know the effect of 8-(4-chlorophenylthio) cyclic 3 5 adenosine monophosphate (cpt-cAMP: 5000 ng/ml) on selected biochemical parameters in Sertoli cells isolated from testis of mice over-expressing bovine growth hormone. The results indicated that cpt-cAMP treated Sertoli cells isolated from mice over-expressing bGH showed a significant increase (p<0.001) in the levels of total proteins (33.78%), DNA (167.81%), RNA (152.38%) and lactic acid (49.56%) levels as compared to treated normal Sertoli cells over a period of five days. However, surprisingly, cpt-cAMP-induced effects in Sertoli cells isolated from testis of transgenic and normal mice were significantly lower as compared to their respective controls. From the results, it can be concluded that cpt-cAMP-induced changes in selected biochemical variables might indicate reversal effects against GH-regulation of protein synthesis and lactate production. These results provide basic information related to the response of GH against excess levels of second messengers in the regulation of metabolic fate of Sertoli cells.

Regulation of Lipid Signaling by Diacylglycerol Kinases during T Cell Development and Function.

Diacylglycerol (DAG) and phosphatidic acid (PA) are bioactive lipids synthesized when the T cell receptor binds to a cognate peptide-MHC complex. DAG triggers signaling by recruiting Ras guanyl-releasing protein 1, PKCθ, and other effectors, whereas PA binds to effector molecules that include mechanistic target of rapamycin, Src homology region 2 domain-containing phosphatase 1, and Raf1. While DAG-mediated pathways have been shown to play vital roles in T cell development and function, the importance of PA-mediated signals remains less clear. The diacylglycerol kinase (DGK) family of enzymes phosphorylates DAG to produce PA, serving as a molecular switch that regulates the relative levels of these critical second messengers. Two DGK isoforms, α and ζ, are predominantly expressed in T lineage cells and play an important role in conventional αβ T cell development. In mature T cells, the activity of these DGK isoforms aids in the maintenance of self-tolerance by preventing T cell hyper-activation and promoting T cell anergy. In this review, we discuss the roles of DAG-mediated pathways, PA-effectors, and DGKs in T cell development and function. We also highlight recent work that has uncovered previously unappreciated roles for DGK activity, for instance in invariant NKT cell development, anti-tumor and anti-viral CD8 responses, and the directional secretion of soluble effectors.

Mitochondrial DNA Oxidative Damage and Repair in Aging and Alzheimer's Disease

Mitochondria are fundamental to the life and proper functioning of cells. These organelles play a key role in energy production, in maintaining homeostatic levels of second messengers (e.g., reactive oxygen species and calcium), and in the coordination of apoptotic cell death. The role of mitochondria in aging and in pathophysiological processes is constantly being unraveled, and their involvement in neurodegenerative processes, such as Alzheimer's disease (AD), is very well known. A considerable amount of evidence points to oxidative damage to mitochondrial DNA (mtDNA) as a determinant event that occurs during aging, which may cause or potentiate mitochondrial dysfunction favoring neurodegenerative events. Concomitantly to reactive oxygen species production, an inefficient mitochondrial base excision repair (BER) machinery has also been pointed to favor the accumulation of oxidized bases in mtDNA during aging and AD progression. The accumulation of oxidized mtDNA bases during aging increases the risk of sporadic AD, an event that is much less relevant in the familial forms of the disease. This aspect is critical for the interpretation of data arising from tissue of AD patients and animal models of AD, as the major part of animal models rely on mutations in genes associated with familial forms of the disease. Further investigation is important to unveil the role of mtDNA and BER in aging brain and AD in order to design more effective preventive and therapeutic strategies.

Effects of Parathyroid Hormone on Functional Activity of the Synaptosomes

We examined the effects of parathyroid hormone (PTH) applied in physiological doses on the entry of 45Ca2+, release of 3H-GABA, and also on the contents of 1,2-diacylglycerol (DAG) and cAMP in preparations of synaptosomes obtained from the brain cortex of mongrel albino rats. It was found that PTH intensified 45Ca2+ entry in the synaptosomes. Potassium depolarization of the synaptosomal membranes did not considerably influence the entry of these ions under the influence of PTH. At the same time, evoked 45Ca2+ entry was greater than the spontaneous one in control preparations. Spontaneous release of 3H-GABA increased under the influence of PTH. At the same time, there was no additional release of 3H-GABA under depolarization conditions. Applications of PTH increased the contents of cAMP and DAG in the examined nerve terminals. It is supposed that increases in the levels of the above messengers predetermine activation of the corresponding protein kinases and phosphorylation of membrane proteins. This, finally, leads to realization of the hormonal signal, namely intensified release of the neurotransmitter.

Inverse correlation of intracellular calcium and cyclic AMP levels in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common renal tumour in adults. Altered levels of secondary messengers, that is, intracellular calcium and cyclic AMP (cAMP), have been implicated in the pathogenesis of various malignancies. In the present study, we measured levels of intracellular calcium and cAMP in RCC. The intracellular calcium level was significantly reduced, whereas the cAMP level was significantly augmented in RCC as compared with adjacent grossly normal renal parenchyma.

Zinc sulphate attenuates chloride secretion in Human colonic mucosae in vitro

Zinc's usefulness in the treatment of diarrhoea is well established as an addition to oral rehydration. Mechanisms of action of zinc have been explored in intestinal epithelia from rodents and in cell lines. The aim was to examine how zinc alters ion transport and signal transduction in human colon in vitro. Voltage clamped colonic sheets obtained at the time of surgical resection were used to quantify ion transport responses to established secretagogues. Nystatin permeabilisation was used to study basolaterally-sited ion channels. Direct actions of zinc were determined using preparations of colonic crypts isolated from human mucosal sheets. Electrophysiological measurements revealed zinc to be an inhibitor of electrogenic ion transport stimulated by forskolin, PGE2, histamine and carbachol in isolated human colonic epithelium. Basolateral addition of zinc sulphate had no direct effect on the epithelium. To further outline the mechanism of action, levels of secondary intracellular messengers (3’, 5’-cyclic adenosine monophosphate; cAMP) were determined in isolated colonic crypts, and were found to be reduced by zinc sulphate. Finally, indirect evidence from nystatin-permeabilised mucosae further suggested that zinc inhibits basolateral K+ channels, which are critical for transepithelial Cl− secretion linked to water flux. Anti-secretory, and therefore anti-diarrhoeal, actions of exogenous zinc are due, at least in part, to direct basolateral epithelial K+ channel inhibition.

Connecting type VI secretion, quorum sensing, and c-di-GMP production in fish pathogen Vibrio alginolyticus through phosphatase PppA

Vibrio alginolyticus, a Gram-negative marine bacterium, has brought about severe economic damage to the mariculture industry by causing vibriosis in various fish species. We are intrigued in the regulation of the pathogenesis in this bacterium. Here, we reported a complex regulatory connection among the newly defined type VI secretion system (T6SS), quorum sensing (QS), and 3′,5′-cyclic diguanylic acid (c-di-GMP) signal through the phosphatase PppA encoded in the T6SS gene cluster of V. alginolyticus. Whole-genome transcriptome analysis revealed various regulatory targets of PppA including the T6SS substrate hemolysin coregulated protein (Hcp), quorum sensing regulator LuxR, exotoxin alkaline serine protease (Asp), flagellar proteins, as well as proteins involved in polysaccharide biosynthesis and transport. Western blot analysis showed PppA served as a negative regulator of the expression and secretion of Hcp1. Mutation of pppA resulted in an increased level of the intracellular second messenger c-di-GMP and a decreased expression of the QS regulator LuxR as well as exotoxin Asp. Complementation of intact pppA gene in ΔpppA mutant restored the production of c-di-GMP, LuxR, and Asp to the wild-type level. Phenotypic studies suggested that PppA takes part in the modulation of biofilm formation, motility, and cell aggregation. These results demonstrated new roles of PppA in controlling virulence factors and pleiotropic phenotypes and contributed to our understanding of the regulation of pathogenesis in V. alginolyticus.

Diet, Obesity, and Prostate Health: Are We Missing the Link?

Prostate problems, such as benign prostatic hyperplasia, prostatic intra-epithelial neoplasia, prostatitis, and prostate cancer have been recognized as problems largely related to androgens and genetic factors. They affect a large fraction of the elderly population, contributing significantly to morbidity and mortality. Estrogen has also now been recognized as one of the important regulators of prostate growth. Diet, general health, and obesity were disregarded as the causative or complicating factors until very recently. Increasing episodes of prostate problems, complications in overweight/obese individuals, or both have attracted attention toward these contemporary risk factors. Prostate problems are reportedly less frequent or less severe in areas in which a plant-based diet is predominant. Consumption of certain fatty acids, particularly of animal origin, has been correlated with increased prostate problems. As adipose tissue is increasingly being regarded as hormonally active tissue, high body fat and obesity need in-depth exploration to understand the associated risk of prostate problems. Adipose tissue is now known to affect circulating levels of several bioactive messengers and therefore could affect the risk of developing prostate problems in addition to several other well-recognized health problems. Nevertheless, increased plasma volume, excess tissue growth, and fat deposition could affect resection and number of biopsies required, thus adding further complications because of a delayed diagnosis. In short, evidence is gathering to support the influence of diet and obesity on prostate health. In this review article, we have tried to make this connection more apparent using supporting published data.

A mitochondrial flirtation with the nucleus

The subcellular distribution of mitochondria may influence cellular functions by controlling the local levels of second messengers including reactive oxygen species (ROS). Al-Mehdi et al. describe how perinuclear clustering of mitochondria can regulate hypoxia-induced gene expression. Hypoxia was found to trigger microtubule-dependent accumulation of mitochondria near the nucleus in pulmonary artery endothelial cells. This correlated with high nuclear ROS levels and oxidative modifications at the VEGF (vascular endothelial growth factor) promoter. The resulting increase in binding of hypoxia-induced factor 1α (HIF1α) to the VEGF promoter and in VEGF mRNA levels suggested that mitochondrial localization can regulate hypoxia-induced gene expression.

The YfiBNR Signal Transduction Mechanism Reveals Novel Targets for the Evolution of Persistent Pseudomonas aeruginosa in Cystic Fibrosis Airways

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

Model for Glucagon Secretion by Pancreatic α-Cells

Glucagon hormone is synthesized and released by pancreatic α-cells, one of the islet-cell types. This hormone, along with insulin, maintains blood glucose levels within the physiological range. Glucose stimulates glucagon release at low concentrations (hypoglycemia). However, the mechanisms involved in this secretion are still not completely clear. Here, using experimental calcium time series obtained in mouse pancreatic islets at low and high glucose conditions, we propose a glucagon secretion model for α-cells. Our model takes into account that the resupply of releasable granules is not only controlled by cytoplasmic , as in other neuroendocrine and endocrine cells, but also by the level of extracellular glucose. We found that, although calcium oscillations are highly variable, the average secretion rates predicted by the model fall into the range of values reported in the literature, for both stimulated and non-stimulated conditions. For low glucose levels, the model predicts that there would be a well-controlled number of releasable granules refilled slowly from a large reserve pool, probably to ensure a secretion rate that could last for several minutes. Studying the α-cell response to the addition of insulin at low glucose, we observe that the presence of insulin reduces glucagon release by decreasing the islet level. This observation is in line with previous work reporting that dynamics, mainly frequency, is altered by insulin [1]. Thus, the present results emphasize the main role played by and glucose in the control of glucagon secretion by α-cells. Our modeling approach also shows that calcium oscillations potentiate glucagon secretion as compared to constant levels of this cellular messenger. Altogether, the model sheds new light on the subcellular mechanisms involved in α-cell exocytosis, and provides a quantitative predictive tool for studying glucagon secretion modulators in physiological and pathological conditions.

The Bacterial Stressosome: A Modular System that Has Been Adapted to Control Secondary Messenger Signaling

The stressosome complex regulates downstream effectors in response to environmental signals. In Bacillus subtilis, it activates the alternative sigma factor σ(B), leading to the upregulation of the general stress regulon. Herein, we characterize a stressosome-regulated biochemical pathway in Moorella thermoacetica. We show that the presumed sensor, MtR, and the scaffold, MtS, form a pseudo-icosahedral structure like that observed in B.subtilis. The N-terminal domain of MtR is structurally homologous to B.subtilis RsbR, despite low sequence identity. The affinity of the switch kinase, MtT, for MtS decreases following MtS phosphorylation and not because of structural reorganization. Dephosphorylation of MtS by the PP2C type phosphatase MtX permits the switch kinase to rebind the stressosome to reset the response. We also show that MtT regulates cyclic di-GMP biosynthesis through inhibition of a GG(D/E)EF-type diguanylate cyclase, demonstrating that secondary messenger levels are regulated by the stressosome.