Global Regulator MorA Affects Virulence-Associated Protease Secretion in Pseudomonas aeruginosa PAO1

Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors – surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.     

Atomic force microscopy (AFM) imaging suggests that stromal interaction molecule 1 (STIM1) binds to Orai1 with sixfold symmetry

Depletion of Ca²⁺ from the endoplasmic reticulum (ER) lumen triggers the opening of Ca²⁺ release-activated Ca²⁺ (CRAC) channels at the plasma membrane. CRAC channels are activated by stromal interaction molecule 1 (STIM1), an ER resident protein that senses Ca²⁺ store depletion and interacts with Orai1, the pore-forming subunit of the channel. The subunit stoichiometry of the CRAC channel is controversial. Here we provide evidence, using atomic force microscopy (AFM) imaging, that Orai1 assembles as a hexamer, and that STIM1 binds to Orai1 with sixfold symmetry. STIM1 associates with Orai1 in the form of monomers, dimers, and multimeric string-like structures that form links between the Orai1 hexamers. Our results provide new insights into the nature of the interactions between STIM1 and Orai1.     

<Emphasis Type="Italic">Acr</Emphasis><Emphasis Type="Italic">emonium</Emphasis> Species: A Review of the Etiological Agents of Emerging Hyalohyphomycosis

Unusual fungal agents that exist environmentally as saprophytes can often lead to opportunistic infections. Hyalohyphomycosis is a group of fungal infections caused by fungi characterized by hyaline septate hyphae and can infect both immunocompetent as well as immunocompromised patients. Many a times it becomes difficult to distinguish a pathogenic and a contaminant fungus, because many such agents can assume clinical significance depending on circumstances. Subcutaneous and invasive fungal infection due to the emerging hyalohyphomycotic fungus, Acremonium, has drawn the attention of clinicians and microbiologists, as a potential pathogen in patients with and without underlying risk factors. Generally considered to be minimally invasive in the past, genus Acremonium has been responsible for eumycotic mycetomas and focal infections in otherwise healthy individuals. It has also been increasingly implicated in systemic fungal diseases. The management with different antifungals in various clinical situations has been very conflicting and hence needs to be carefully evaluated. This overview is an endeavor to consolidate the available clinical infections due to Acremonium and the recommendations on treatment.     

Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-β effectors

Smad1 is a downstream effector of the BMP signaling pathway that binds regulatory DNA to execute gene expression programs leading to, for example, the maintenance of pluripotency in mice. On the contrary, the TGF-β-activated Smad3 triggers strikingly different programs such as mesodermal differentiation in early development. Because Smad1 and Smad3 contain identical amino acids at the DNA contact interface it is unclear how they elicit distinctive bioactivities. Here, we report the crystal structure of the MH1 domain of Smad1 bound to a palindromic Smad binding element. Surprisingly, the DNA contact interface of Smad1 is drastically rearranged when compared to Smad3. The N-terminal helix 1 of Smad1 is dislodged from its intramolecular binding site and adopts a domain swapped arrangement with a symmetry-related molecule. As a consequence, helix 2 kinks away from the double helix disabling several key phosphate backbone interactions. Thermal melting analysis corroborates a decompacted conformation of Smad1 and DNA binding assays indicate a lower overall affinity of Smad1 to DNA but increased cooperativity when binding to palindromic DNA motifs. These findings suggest that Smad1 and Smad3 evolved differential qualities to assemble on composite DNA elements and to engage in co-factor interactions by remodeling their N-termini.     

Surgical removal of duct occluder device under mild hypothermia without cardiopulmonary bypass

Because the use of percutaneous intervention is increasing for the closure of the patent ductus arteriosus, the procedure-related complications are also on rise, with migration of the device being most common. The routine practice is to remove the migrated duct occluder device under cardiopulmonary bypass. Amplatzer duct occluder used in a 4-month-old infant dislodged into the descending thoracic aorta. It was removed by the posterolateral thoracotomy under mild hypothermia through juxtaductal aortotomy between the aortic cross-clamps. The use of cardiopulmonary bypass is thus avoided.     

<Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis> (Fenugreek) Protects Against Selenite-Induced Oxidative Stress in Experimental Cataractogenesis

Cataract is the opacification in eye lens and leads to 50% of blindness worldwide. The present study was undertaken to evaluate the anticataract potential of Trigonella foenum-graecum Linn seeds (fenugreek) in selenite-induced in vitro and in vivo cataract. In vitro enucleated rat lenses were maintained in organ culture containing Dulbecco’s modified Eagles medium (DMEM) alone or in addition with 100 μM selenite and served as the normal and control groups, respectively. For the test group, the medium was supplemented with selenite and T. foenum-graecum aqueous extract. The lenses were incubated for 24 h at 37°C. After incubation, the lenses were processed for the estimation of reduced glutathione (GSH), lipid peroxidation product (malondialdehyde), and the antioxidant enzymes. In vivo selenite cataract was induced in 9-day-old rats by subcutaneous injection of sodium selenite (25 μmol/kg body weight). Animals in the test group were injected with different doses of aqueous extract of T. foenum-graecum 4 h before the selenite challenge. A fall in GSH and a rise in malondialdehyde levels were observed in control as compared to normal lenses. T. foenum-graecum significantly (P < 0.01) restored glutathione and decreased malondialdehyde levels. A significant restoration in the activities of antioxidant enzymes such as superoxide dismutase (P < 0.01), catalase, (P < 0.01), glutathione peroxidase (P < 0.01), and glutathione-S-transferase (P < 0.01) was observed in the T. foenum-graecum supplemented group as compared to control. In vivo, none of the eyes was found with nuclear cataract in treated group as opposed to 72.5% in the control group. T. foenum-graecum protects against experimental cataract by virtue of its antioxidant properties. Further studies are warranted to explore its role in human cataract.     

A multiattribute utility evaluation of different methods for the detection of enteric protozoa causing diarrhea in AIDS patients

Background  

Enteric protozoa and sporozoa have emerged as important opportunistic parasites and can cause fatal infections in AIDS patients. The line of treatment being different for them necessitates an accurate and prompt identification of these to avoid empirical treatment. In this study which is the first of its kind from India we did a comprehensive evaluation of different techniques, comparing them on the basis of the attributes like yield, cost, time taken, expertise and infrastructure. For the first time combination of Calcoflour White and DAPI, a nuclear stain, were used to identify Microsporidia spp. Thus, a diagnostic protocol was devised for rapid, sensitive and cost effective identification of the opportunistic enteric protozoa.     

Effect of sequence hydrophobicity and bilayer width upon the minimum length required for the formation of transmembrane helices in membranes

The minimum hydrophobic length necessary to form a transmembrane (TM) helix in membranes was investigated using model membrane-inserted hydrophobic helices. The fluorescence of a Trp at the center of the sequence and its sensitivity to quenching were used to ascertain helix position within the membrane. Peptides with hydrophobic cores composed of poly(Leu) were compared to sequences containing a poly 1:1 Leu:Ala core (which have a hydrophobicity typical of natural TM helices). Studies varying bilayer width revealed that the poly(Leu) core peptides predominately formed a TM state when the bilayer width exceeded hydrophobic sequence length by (i.e. when negative mismatch was) up to ∼ 11-12 Å (e.g. the case of a 11-12 residue hydrophobic sequence in bilayers with a biologically relevant width, i.e. dioleoylphosphatidylcholine (DOPC) bilayers), while poly(LeuAla) core peptides formed predominantly TM state with negative mismatch of up to 9 Å (a 13 residue hydrophobic sequence in DOPC bilayers). This indicates that minimum length necessary to form a predominating amount of a TM state (minimum TM length) is only modestly hydrophobicity-dependent for the sequences studied here, and a formula that defines the minimum TM length as a function of hydrophobicity for moderately-to-highly hydrophobic sequences was derived. The minimum length able to form a stable TM helix for alternating LeuAla sequences, and that for sequences with a Leu block followed by an Ala block, was similar, suggesting that a hydrophobicity gradient along the sequence may not be an important factor in TM stability. TM stability was also similar for sequences flanked by different charged ionizable residues (Lys, His, Asp). However, ionizable flanking residues destabilized the TM configuration much more when charged than when uncharged. The ability of short hydrophobic sequences to form TM helices in membranes in the presence of substantial negative mismatch implies that lipid bilayers have a considerable ability to adjust to negative mismatch, and that short TM helices may be more common than generally believed. Factors that modulate the ability of bilayers to adjust to mismatch may strongly affect the configuration of short hydrophobic helices.