Structure and membrane-targeting of a Bordetella pertussis effector N-terminal domain

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Like other cytotoxicity-mediating effectors, BteA uses its multifunctional N-terminal domain to target phosphatidylinositol (PI)-rich microdomains in the host membrane. Despite their structural similarity, T3SS effectors exhibit a variable range of membrane interaction modes, and currently only limited structural information is available for the BteA membrane-targeting domain and the molecular mechanisms underlying its function. Employing a synergistic combination of structural methods, here we determine the structure of this functional domain and uncover key molecular determinants mediating its interaction with membranes. Residues 29–121 of BteA form an elongated four-helix bundle packed against two shorter perpendicular helices, the second of which caps the domain in a critical ‘tip motif’. A flexible region preceding the BteA helical bundle contains the characteristic β-motif required for binding its cognate chaperone BtcA. We show that BteA targets PI(4,5)P₂-containing lipoprotein nanodiscs and binds a soluble PI(4,5)P₂ analog via an extensive positively charged surface spanning its first two helices, and that this interaction is weaker for PI(3,5)P₂ and abolished for PI(4)P. We confirmed this model of membrane-targeting by observation of BteA-induced changes in the structure of PI(4,5)P₂-containing phospholipid bilayers using small-angle X-ray scattering (SAXS). We also extended these results to a larger BteA domain (residues 1–287), confirming its interaction with bilayers using calorimetry, fluorescence and SAXS methods. This novel view of the structural underpinnings of membrane targeting by BteA is an important step towards a comprehensive understanding of cytotoxicity in Bordetella, as well as interactions of a broad range of pathogens with their respective hosts.     

Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression

The mechanisms by which excessive glucocorticoids cause muscular atrophy remain unclear. We previously demonstrated that dexamethasone increases the expression of myostatin, a negative regulator of skeletal muscle mass, in vitro. In the present study, we tested the hypothesis that dexamethasone-induced muscle loss is associated with increased myostatin expression in vivo. Daily administration (60, 600, 1,200 micro g/kg body wt) of dexamethasone for 5 days resulted in rapid, dose-dependent loss of body weight (-4.0, -13.4, -17.2%, respectively, P < 0.05 for each comparison), and muscle atrophy (6.3, 15.0, 16.6% below controls, respectively). These changes were associated with dose-dependent, marked induction of intramuscular myostatin mRNA (66.3, 450, 527.6% increase above controls, P < 0.05 for each comparison) and protein expression (0.0, 260.5, 318.4% increase above controls, P < 0.05). We found that the effect of dexamethasone on body weight and muscle loss and upregulation of intramuscular myostatin expression was time dependent. When dexamethasone treatment (600 micro g. kg-1. day-1) was extended from 5 to 10 days, the rate of body weight loss was markedly reduced to approximately 2% within this extended period. The concentrations of intramuscular myosin heavy chain type II in dexamethasone-treated rats were significantly lower (-43% after 5-day treatment, -14% after 10-day treatment) than their respective corresponding controls. The intramuscular myostatin concentration in rats treated with dexamethasone for 10 days returned to basal level. Concurrent treatment with RU-486 blocked dexamethasone-induced myostatin expression and significantly attenuated body loss and muscle atrophy. We propose that dexamethasone-induced muscle loss is mediated, at least in part, by the upregulation of myostatin expression through a glucocorticoid receptor-mediated pathway.     

Delayed Wound Healing in Heat Stable Antigen (HSA/CD24)-Deficient Mice

Background

Healthy individuals rarely have problems with wound healing. Most skin lesions heal rapidly and efficiently within one to two weeks. However, many medical and surgical complications can be attributed to deficiencies in wound repair. Open wounds have lost the barrier that protects tissues from bacterial invasion and allows the escape of vital fluids. Without expeditious healing, infections become more frequent. The CD24 gene encodes a heavily-glycosylated cell surface protein anchored to the membrane by phosphatidylinositol. CD24 plays an important role in the adaptive immune response and controls an important genetic checkpoint for homeostasis and autoimmune diseases in both mice and humans. We have previously shown that overexpression of CD24 results in increased proliferation and migration rates.

Aim

To examine the role of CD24 in the wound healing process.

Methods

An excisional model of wound healing was used and delayed wound healing was studied in genetically modified heat stable antigen (HSA/CD24)-deficient mice (HSA ^-/-) compared to wild-type (WT) mice.

Results

Large full-thickness skin wounds, excised on the back of mice, exhibited a significant delay in the formation of granulation tissue, and in wound closure when compared to their WTHSA ^+/+ littermates. Wounds were histologically analyzed and scored, based on the degree of cellular invasion, granulation tissue formation, vascularity, and re-epithelialization. Additionally, in stitched wounds, the HSA ^-/- mice failed to maintain their stitches; they did not hold and fell already 24 hours, revealing erythematous wound fields. Re-expression of HSA, delivered by lentivirus, restored the normal healing phenotype, within 24 hours post-injury, and even improved the healing in WT, and in BalbC mice.

Conclusions

Delayed wound-healing in the absence of HSA/CD24 suggests that CD24 plays an important role in this process. Increased expression of CD24, even in the normal state, may be used to enhance wound repair.     

Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae

Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents for treating cardiovascular obstructions such as stroke. Glycoprotein rt-PA is a serine protease, consisting of 527 amino acids of which 35 are cysteine residues. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae has been considered because of its safety aspects and special attributes in expression of complex proteins. In this study, two expression cassettes, each one including two copies of t-PA cDNA, were used for integration into the L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Expression of active rt-PA was confirmed by Western blot and Zymography tests. Real-time PCR analysis was applied to investigate the presence of multiple t-PA gene copies in the parasite genome. Correlation of t-PA gene dosage and production rate was confirmed with real-time PCR. It was shown that the expression level of rt-PA in L. tarentolae is at least 480 IU/mL of culture media. This concentration of rt-PA is seven times higher than what was reported in previous studies in L. tarentolae and some other eukaryotic systems.     

Molecularly imprinted polymer cartridges coupled on-line with high performance liquid chromatography for simple and rapid analysis of dextromethorphan in human plasma samples

In this paper, a novel method is described for automated determination of dextromethorphan in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as a sample clean-up technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and dextromethorphan as template molecule. These imprinted polymers were used as solid-phase extraction sorbent for the extraction of dextromethorphan from human plasma samples. Various parameters affecting the extraction efficiency of the MIP cartridges were evaluated. The high selectivity of the sorbent coupled to the high performance liquid chromatographic system permitted a simple and rapid analysis of this drug in plasma samples with limits of detection (LOD) and quantification (LOQ) of 0.12 ng/mL and 0.35 ng/mL, respectively. The MIP selectivity was evaluated by analyzing of the dextromethorphan in presence of several substances with similar molecular structures and properties. Results from the HPLC analyses showed that the recoveries of dextromethorphan using MIP cartridges from human plasma samples in the range of 1-50 ng/mL were higher than 87%.     

The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.     

Schwartz Formula: Is One k-Coefficient Adequate for All Children?

Background/Objective

Plasma-creatinine-based equations to estimate the glomerular filtration rate are recommended by several clinical guidelines. In 2009, Schwartz et al. adapted the traditional Schwartz equation to children and adolescents but did not find different k-coefficients between children and adolescents (k = 36.5 for all patients). We reevaluated the coefficient of the 2009-Schwartz formula according to sex and age in a pediatric population.

Patients/Methods

We used linear mixed-effects models to reestimate the 2009-Schwartz k-coefficient in 360 consecutive French subjects aged 1 to 18 years referred to a single centre between July 2003 and July 2010 (965 measurements). We assessed the agreement between the estimated glomerular filtration rate obtained with the new formula (called Schwartz-Lyon) and the rate measured by inulin clearance. We then compared this agreement to the one between the measured glomerular filtration rate and 2009-Schwartz formula, first in the French then in a Swedish cohort.

Results

In Schwartz-Lyon formula, k was estimated at 32.5 in boys <13 years and all girls and at 36.5 in boys aged ≥13 years. The performance of this formula was higher than that of 2009-Schwartz formula in children <13 years. This was first supported by a statistically significant reduction of the overestimation of the measured glomerular filtration rate in both cohorts, by better 10% and 30% accuracies, and by a better concordance correlation coefficient.

Conclusions

The performance and simplicity of Schwartz formula are strong arguments for its routine use in children and adolescents. The specific coefficient for children aged <13 years further improves this performance.     

Analysis of Antarctic proteobacteria by PCR fingerprinting and screening for antimicrobial secondary metabolites

Fifty-seven proteobacterium species were successfully isolated from soils of Barrientos Island of the Antarctic using 11 different isolation media. Analysis of 16S rDNA sequencing of these isolates showed that they belonged to eight different genera, namely Bradyrhizobium, Sphingomonas, Methylobacterium, Caulobacter, Paracoccus, Ralstonia, Rhizobium, and Staphylococcus. All isolates were studied for capability of producing antimicrobial and antifungal secondary metabolites using high-throughput screening models. Approximately 23 (13/57) and 2% (1/57) of isolates inhibited growth of Candida albicans ATCC 10231(T) and Staphylococcus aureus ATCC 51650(T), respectively. These results indicated that proteobacterium species isolates from Antarctic could serve as potential source of useful bioactive metabolites. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting produced nine clusters and 13 single isolates, with a high D value of 0.9248. RAPD fingerprinting produced six clusters and 13 single isolates, with a relatively low D value of 0.7776. ERIC-PCR analysis proved to have better discrimination capability than RAPD analysis and generated better clustering for all proteobacterium species isolates. We conclude that ERIC-PCR is a robust, reliable and rapid molecular typing method for discriminating different genera of proteobacteria.     

Identification of actinomycete communities in Antarctic soil from Barrientos Island using PCR-denaturing gradient gel electrophoresis

The diversity of specific bacteria taxa, such as the actinomycetes, has not been reported from the Antarctic island of Barrientos. The diversity of actinomycetes was estimated with two different strategies that use PCR-denaturing gradient gel electrophoresis. First, a PCR was applied, using a group-specific primer that allows selective amplification of actinomycete sequences. Second, a nested-PCR approach was used that allows the estimation of the relative abundance of actinomycetes within the bacterial community. Molecular identification, which was based on 16S rDNA sequence analysis, revealed eight genera of actinomycetes, Actinobacterium, Actinomyces, an uncultured Actinomycete, Streptomyces, Leifsonia, Frankineae, Rhodococcus, and Mycobacterium. The uncultured Actinomyces sp and Rhodococcus sp appear to be the prominent genera of actinomycetes in Barrientos Island soil. PCR-denaturing gradient gel electrophoresis patterns were used to look for correlations between actinomycete abundance and environmental characteristics, such as type of rookery and vegetation. There was a significant positive correlation between type of rookery and abundance of actinomycetes; soil samples collected from active chinstrap penguin rookeries had the highest actinomycete abundance. Vegetation type, such as moss, which could provide a microhabitat for bacteria, did not correlate significantly with actinomycete abundance.