Molecular features of heterogeneous vancomycin-intermediate Staphylococcus aureus strains isolated from bacteremic patients

Background  

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) bacteremia is an emerging infection. Our objective was to determine the molecular features of hVISA strains isolated from bacteremic patients and to compare them to methicillin resistant S. aureus (MRSA) and methicillin sensitive S. aureus (MSSA) blood isolates.     

Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Background

Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.

Methodology/Principal Findings

Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.

Conclusions/Significance

These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.     

ProtoNet: hierarchical classification of the protein space

The ProtoNet site provides an automatic hierarchical clustering of the SWISS-PROT protein database. The clustering is based on an all-against-all BLAST similarity search. The similarities' E-score is used to perform a continuous bottom-up clustering process by applying alternative rules for merging clusters. The outcome of this clustering process is a classification of the input proteins into a hierarchy of clusters of varying degrees of granularity. ProtoNet (version 1.3) is accessible in the form of an interactive web site at http://www.protonet.cs.huji.ac.il. ProtoNet provides navigation tools for monitoring the clustering process with a vertical and horizontal view. Each cluster at any level of the hierarchy is assigned with a statistical index, indicating the level of purity based on biological keywords such as those provided by SWISS-PROT and InterPro. ProtoNet can be used for function prediction, for defining superfamilies and subfamilies and for large-scale protein annotation purposes.     

Molecular and Structural Characterization of the HMP-AB Gene Encoding a Pore-Forming Protein from a Clinical Isolate of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

The major outer membrane protein of Acinetobacter baumannii is the heat-modifiable protein HMP-AB, a porin with a large pore size allowing the penetration of solutes having a molecular weight of up to approximately 800 Da. Cross-linking experiments with glutardialdehyde failed to show any cross-linking between the monomers, a fact that proves again that this porin protein functions as a monomeric porin. The specific activity of this porin was found to be similar to that of other monomeric porins. Tryptic digestion of the outer membrane yielded a 23-kDa fragment of the HMP-AB protein that was resistant to further trypsin treatment. This observation indicates that HMP-AB is assembled in the membrane in a manner similar to monomeric porins. Cloning of the HMP-AB gene revealed an open reading frame of 1038 bp encoding a protein of 346 amino acids and a calculated molecular mass of 35,636 Da. The amino acid sequence and composition were typical of Gram-negative bacterial porins: a highly negative hydropathy index, absence of hydrophobic residue stretches, a slightly negative total charge, low instability index, high glycine content, and an absence of cysteine residues. Sequence comparison of HMP-AB with other outer membrane proteins revealed a clear homology with the monomeric outer membrane proteins, outer membrane protein A (OmpA) of Enterobacteria, and outer membrane protein F (OprF) of Pseudomonas sp. Secondary structure analysis indicated that HMP-AB has a 172-amino acid N-terminal domain that spans the outer membrane by eight amphiphilic beta strands and a C-terminal domain that apparently serves as an anchoring protein to the peptidoglycan layer. The results also indicate that HMP-AB belongs to the eight transmembrane beta-strand family of outer membrane proteins.     

Cloning and Characterization of the Gene Encoding for OMP-PD Porin: The Major <Emphasis Type="Italic">Photobacterium damsela</Emphasis> Outer Membrane Protein

The outer membrane protein of Photobacterium damsela (OMP-PD) and the gene encoding for this porin protein were isolated and characterized. The deduced amino acid sequence of the OMP-PD monomer has 338 amino acids and a calculated molecular weight of 36,951 Da. This sequence includes a 22-amino acid signal peptide at the N-terminal, which is not found when the monomer is located in the outer membrane. Native OMP-PD protein forms a trimeric structure of approximately 110 kDa. It exhibits resistance to proteases, and it can be cleaved only following denaturation by SDS. The degree of identity of the OMP-PD amino acid sequence to porins from the Enterobacteriaceae was only 24%. Identity to Vibrio or Photobacterium porins was 38% and 48%, respectively. Nevertheless, the multiple alignment of this sequence with other structurally defined Enterobacteria porins demonstrated that the location of the 16 beta-strands and eight external loops, including a larger external L3 loop, are conserved in OMP-PD. These results, together with the previously known ability of OMP-PD to form an ion channel in artificial liposomes, strongly support its role as a porin in P. damsela and will help further investigations into the role of OMP-PD in P. damsela pathogenicity.     

Growth-inhibitory effect of hemin on staphylococci

The antibacterial activity of hemin onStaphylococcus aureus is described. Hemin binding to bacteria was a rapid process, and each cell accumulated 5×10⁵ to 1×10⁶ molecules within 5 min. Bacterial growth was stopped completely after 30 min from addition of low concentration of hemin (3–10 g/ml). Cell viability was reduced by 99.9% in 1 h of exposure, and the effect was consistent at any stage of the growth curve whenever hemin was added. Glucose utilization was arrested immediately after hemin addition, and no CO₂ was produced. The survivors of hemin treatment regrow in a time-related kinetics depending on the dose of hemin to which the cells were exposed. The recovered bacteria were again sensitive to hemin, similar to an untreated culture. We suggested that the recovery phenomenon is a result of an on-off mechanism regulating sensitivity to hemin, rather than a selection mechanism giving rise to hemin-resistant mutants.     

Inactivation of anaerobic bacteria by various photosensitized porphyrins or by hemin

The photodynamic effects of deuteroporphyrin (DP), hematoporphyrin derivative (HPD), hematoporphyrin (HP), or protoporphyrin (PP) on a variety of anaerobic microorganisms were examined in this study. The majority of the species, among the 350 strains tested, were inhibited by concentrations of 2.5 g/ml of light-activated DP. Species found to be resistant to this treatment includedBilophila wadsworthia, Fusobacterium mortiferum, Fusobacterium varium, andBacteroides gracilis. These species were inhibited by concentrations of >60 g/ml of DP. The porphyrin-producing species,Porphyromonas andPrevotella spp, were all inhibited by 2.5 g/ml DP and light. Comparing the photodynamic activity of the porphyrins used onPorphyromonas strains resulted in the following pattern: DP>HPD>HP>PP.Porphyromonas spp., Gram-positive cocci, and many Gram-positive rods (excluding clostridia) were inactivated by hemin (a metal-containing porphyrin) at 10–20 g/ml. Hemin inhibitory action was not affected by light. Binding and insertion of DP into bacteria (both inactivated and non-inactivated strains by DP and light) were monitored by the characteristic fluorescence band of bound DP at 622 nm.Porphyromonas spp. bound DP tightly, whereas only low binding was seen withB. wadsworthia and other DP-resistant species. High binding of DP toB. wadsworthia can be achieved by pretreatment of the bacteria with imipenem or cefoxitin, -lactam agents known to interfere with the integrity of the cell wall. If cell wall integrity is disturbed (e.g., by these agents), inactivation ofB. wadsworthia by DP can occur.     

Intermediate structures in the cholate-phosphatidylcholine vesicle-micelle transition

The vesicle-micelle transition of egg phosphatidylcholine (PC) and sodium cholate was described by comparing cryo-transmission electron microscopic (cryo-TEM) images of the structures formed to the associated turbidity changes. These experiments were designed to identify the morphology of the intermediates between vesicles and small spheroidal mixed micelles. With increasing cholate concentration, the vesicular structures changed size and more multilamellar vesicles were seen. Between the apparent upper and lower phase boundaries, three structures were observed: open vesicles, large bilayer sheets (twenty to several hundred nanometers in diameter), and long (150-300 nm) flexible cylindrical micelles. The cylindrical micelles evolved from the edges of the bilayer sheets. At higher relative cholate concentration, the phase boundary was sharply defined by optical clarification of the egg PC-cholate mixtures. Cryo-TEM revealed only small spheroidal mixed micelles at this transition. These results provide the first direct evidence of the structural pathway or of molecular intermediates between a lamellar and a micellar state. Understanding these specific intermediates and the transitions between them is essential to developing reconstitution protocols and properly analyzing either activity or structural data obtained from cholate-dispersed membrane proteins.     

Cell encapsulation utilizing PEG-fibrinogen hydrogel supports viability and enhances productivity under stress conditions

Recent advances in the bioengineering field have introduced new opportunities enabling cell encapsulation in three-dimensional (3D) structures using either various natural or synthetic materials. However, such hydrogel scaffolds have not been fully biocompatible for cell cultivation due to the lack of physical stability or bioactivity. Here, we utilized a uniquely fabricated semi-synthetic 3D polyethylene glycol-fibrinogen (PEG-Fb) hydrogel scaffold, which exhibits both high stability and high bioactivity, to encapsulate HEK293 cells for the production of human recombinant acetylcholine esterase (AChE). To examine the beneficial bioactive effect of the PEG-Fb scaffold over 2D surfaces, an experimental system was established to compare the viability, proliferation and AChE secretion of encapsulated cells versus non-encapsulated surface-adherent cells in serum starvation. Our results show that the transfer of surface-adherent HEK293 cells from fully enriched medium with 10% FCS to 0.2% FCS resulted in an eightfold reduction in cell number and a fourfold reduction in AChE production. In contrast, the encapsulated cells were highly viable and about twofold more efficient in AChE production. In addition, they had round morphology with a twofold larger cell diameter, supporting the observation of increased AChE production. These results suggest a role of the PEG-Fb scaffold in providing a supportive microenvironment in reduced serum conditions that enhances encapsulated cell functions, opening new directions to study the implementation of this platform in large-scale pharmaceutical protein production.