Response of Staphylococcus aureus physiology and Agr quorum sensing to low-shear modeled microgravity.

Staphylococcus aureus is commonly isolated from astronauts returning from spaceflight. Previous analysis of omics data from S. aureus low Earth orbit cultures indicated significantly increased expression of the Agr quorum sensing system and its downstream targets in spaceflight samples compared to ground controls. In this current study, the rotary cell culture system (RCCS) was used to investigate the effect of low-shear modeled microgravity (LSMMG) on S. aureus physiology and Agr activity. When cultured in the same growth medium and temperature as the previous spaceflight experiment, S. aureus LSMMG cultures exhibited decreased agr expression and altered growth compared to normal gravity control cultures, which are typically oriented with oxygenation membrane on the bottom of the high aspect rotating vessel (HARV). When S. aureus was grown in an inverted gravity control orientation (oxygenation membrane on top of the HARV), reduced Agr activity was observed relative to both traditional control and LSMMG cultures, signifying that oxygen availability may affect the observed differences in Agr activity. Metabolite assays revealed increased lactate and decreased acetate excretion in both LSMMG and inverted control cultures. Secretomics analysis of LSMMG, control, and inverted control HARV culture supernatants corroborated these results, with inverted and LSMMG cultures exhibiting a decreased abundance of Agr-regulated virulence factors and an increased abundance of proteins expressed in low-oxygen conditions. Collectively, these studies suggest that the orientation of the HARV oxygenation membrane can affect S. aureus physiology and Agr quorum sensing in the RCCS, a variable that should be considered when interpreting data using this ground-based microgravity model.IMPORTANCES. aureus is commonly isolated from astronauts returning from spaceflight and from surfaces within human-inhabited closed environments such as spacecraft. Astronaut health and immune function are significantly altered in spaceflight. Therefore, elucidating the effects of microgravity on S. aureus physiology is critical for assessing its pathogenic potential during long-term human space habitation. These results also highlight the necessity of eliminating potential confounding factors when comparing simulated microgravity model data with actual spaceflight experiments.

Human cyclophilin 40 unravels neurotoxic amyloids.

The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer's disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.

Hsp90 activator Aha1 drives production of pathological tau aggregates.

The microtubule-associated protein tau (MAPT, tau) forms neurotoxic aggregates that promote cognitive deficits in tauopathies, the most common of which is Alzheimer's disease (AD). The 90-kDa heat shock protein (Hsp90) chaperone system affects the accumulation of these toxic tau species, which can be modulated with Hsp90 inhibitors. However, many Hsp90 inhibitors are not blood-brain barrier-permeable, and several present associated toxicities. Here, we find that the cochaperone, activator of Hsp90 ATPase homolog 1 (Aha1), dramatically increased the production of aggregated tau. Treatment with an Aha1 inhibitor, KU-177, dramatically reduced the accumulation of insoluble tau. Aha1 colocalized with tau pathology in human brain tissue, and this association positively correlated with AD progression. Aha1 overexpression in the rTg4510 tau transgenic mouse model promoted insoluble and oligomeric tau accumulation leading to a physiological deficit in cognitive function. Overall, these data demonstrate that Aha1 contributes to tau fibril formation and neurotoxicity through Hsp90. This suggests that therapeutics targeting Aha1 may reduce toxic tau oligomers and slow or prevent neurodegenerative disease progression.

Measurement of Staphylococcus aureus Pigmentation by Methanol Extraction.

Many S. aureus strains produce membrane-associated carotenoid pigments, advantageous secondary metabolites that can alter membrane fluidity, resistance to antimicrobial peptides (AMPs) and act as antioxidants, properties that can impact resistance against aspects of the host innate immune system. Several studies have reported connections between mutations in both regulatory (i.e., alternative sigma factor B) and metabolic (purine biosynthesis, oxidative phosphorylation) genes, and noticeable differences in carotenoid pigmentation. This chapter outlines a simple protocol to quantify cellular pigments using a methanol extraction method.