Modelled-Microgravity Reduces Virulence Factor Production in Staphylococcus aureus through Downregulation of agr-Dependent Quorum Sensing.

Bacterial contamination during space missions is problematic for human health and damages filters and other vital support systems. Staphylococcus aureus is both a human commensal and an opportunistic pathogen that colonizes human tissues and causes acute and chronic infections. Virulence and colonization factors are positively and negatively regulated, respectively, by bacterial cell-to-cell communication (quorum sensing) via the agr (accessory gene regulator) system. When cultured under low-shear modelled microgravity conditions (LSMMG), S. aureus has been reported to maintain a colonization rather than a pathogenic phenotype. Here, we show that the modulation of agr expression via reduced production of autoinducing peptide (AIP) signal molecules was responsible for this behavior. In an LSMMG environment, the S. aureus strains JE2 (methicillin-resistant) and SH1000 (methicillin-sensitive) both exhibited reduced cytotoxicity towards the human leukemia monocytic cell line (THP-1) and increased fibronectin binding. Using S. aureus agrP3::lux reporter gene fusions and mass spectrometry to quantify the AIP concentrations, the activation of agr, which depends on the binding of AIP to the transcriptional regulator AgrC, was delayed in the strains with an intact autoinducible agr system. This was because AIP production was reduced under these growth conditions compared with the ground controls. Under LSMMG, S. aureus agrP3::lux reporter strains that cannot produce endogenous AIPs still responded to exogenous AIPs. Provision of exogenous AIPs to S. aureus USA300 during microgravity culture restored the cytotoxicity of culture supernatants for the THP-1 cells. These data suggest that microgravity does not affect AgrC-AIP interactions but more likely the generation of AIPs.

Protein unfolding under force: crack propagation in a network.

The mechanical unfolding of a set of 12 proteins with diverse topologies is investigated using an all-atom constraint-based model. Proteins are represented as polypeptides cross-linked by hydrogen bonds, salt bridges, and hydrophobic contacts, each modeled as a harmonic inequality constraint capable of supporting a finite load before breaking. Stereochemically acceptable unfolding pathways are generated by minimally overloading the network in an iterative fashion, analogous to crack propagation in solids. By comparing the pathways to those from molecular dynamics simulations and intermediates identified from experiment, it is demonstrated that the dominant unfolding pathways for 9 of the 12 proteins studied are well described by crack propagation in a network.

Atomic force microscopy study of human amylin (20-29) fibrils.

Here we present atomic force microscopy images of the fibrils formed by human amylin(20-29). This peptide is a fragment of the polypeptide amylin, the major proteinaceous component of amyloid deposits found in cases of type-II diabetes mellitus. Our results demonstrate that the amylin(20-29) peptide fragment forms amyloid-like fibrils that display polymorphic structures. Twisting along the axis of fibrils was often observed in fibrils aged for 6 hours but disappeared in mature fibrils aged for longer time periods.

The effect of poly(ethylene glycol) molecular architecture on cellular interaction and uptake of DNA complexes.

The cellular uptake of plasmid DNA complexes with a series of tertiary amine methacrylate-ethylene glycol (DMAEMA-EG) copolymers with various architectures was studied using flow cytofluorometry and laser confocal microscopy. The complexes displayed different rates and extents of cellular interaction and internalisation, depending on the copolymer molecular architecture. In general, introduction of oligo(ethylene glycol) [OEG] or poly(ethylene glycol) [PEG] chains decreased both the interaction and cellular internalisation of the DNA complexes but subtle differences were observed. Two block copolymers, a 'bottle-brush' type DMAEMA-block-OEGMA and a linear DMAEMA-block-PEG copolymer (each containing a total of 45 EG units), displayed similar uptake profiles. In contrast, only relatively low uptake of complexes formed by a comb-type statistical copolymer, DMAEMA-stat-PEGMA, was observed, despite each PEG chain comprising 45 EG units. Similar trends were observed with three cell lines, A549, HepG2 and COS-7. However, the absolute values were cell-dependent, with COS-7 cells displaying both the highest rate and extent of uptake. Studies of the association and uptake of the complexes demonstrated that cell associations generally increased over time, with the uptake level and the time profile depending on the polymer architecture. Confocal microscopy studies confirmed that, with the exception of the poorly transfecting comb-type copolymer, the association of complexes with cells resulted in endocytosis.