Alkyl quinolones mediate heterogeneous colony biofilm architecture that improves community-level survival.

Bacterial communities exhibit complex self-organization that contributes to their survival. To better understand the molecules that contribute to transforming a small number of cells into a heterogeneous surface biofilm community, we studied acellular aggregates, structures seen by light microscopy in Pseudomonas aeruginosa colony biofilms using light microscopy and chemical imaging. These structures differ from cellular aggregates, cohesive clusters of cells important for biofilm formation, in that they are visually distinct from cells using light microscopy and are reliant on metabolites for assembly. To investigate how these structures benefit a biofilm community we characterized three recurrent types of acellular aggregates with distinct geometries that were each abundant in specific areas of these biofilms. Alkyl quinolones (AQs) were essential for the formation of all aggregate types with AQ signatures outside the aggregates below the limit of detection. These acellular aggregates spatially sequester AQs and differentiate the biofilm space. However, the three types of aggregates showed differing properties in their size, associated cell death, and lipid content. The largest aggregate type co-localized with spatially confined cell death that was not mediated by Pf4 bacteriophage. Biofilms lacking AQs were absent of localized cell death but exhibited increased, homogeneously distributed cell death. Thus, these AQ-rich aggregates regulate metabolite accessibility, differentiate regions of the biofilm, and promote survival in biofilms.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen with the ability to cause infection in the immune-compromised. It is well established that P. aeruginosa biofilms exhibit resilience that includes decreased susceptibility to antimicrobial treatment. This work examines the self-assembled heterogeneity in biofilm communities studying acellular aggregates, regions of condensed matter requiring alkyl quinolones (AQs). AQs are important to both virulence and biofilm formation. Aggregate structures described here spatially regulate the accessibility of these AQs, differentiate regions of the biofilm community, and despite their association with autolysis, correlate with improved P. aeruginosa colony biofilm survival.

Exploration of the Potential Role for Aß in Delivery of Extracellular Copper to Ctr1.

Amyloid beta (Aß) peptides are notorious for their involvement in Alzheimer's disease (AD), by virtue of their propensity to aggregate to form oligomers, fibrils, and eventually plaques in the brain. Nevertheless, they appear to be essential for correct neurophysiology on the synaptic level and may have additional functions including antimicrobial activity, sealing the blood-brain barrier, promotion of recovery from brain injury, and even tumor suppression. Aß peptides are also avid copper chelators, and coincidentally copper is significantly dysregulated in the AD brain. Copper (Cu) is released in significant amounts during calcium signaling at the synaptic membrane. Aß peptides may have a role in maintaining synaptic Cu homeostasis, including as a scavenger for redox-active Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aß1-16 and Aß4-16 peptides as well-established non-aggregating models of major Aß species in healthy and AD brains, and the Ctr1-14 peptide as a model for the extracellular domain of the human cellular copper transporter protein (Ctr1). With these model peptides and a number of spectroscopic techniques, we investigated whether the Cu complexes of Aß peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aß1-16 fully and rapidly delivered Cu(II) to Ctr1-14 along the affinity gradient. Such delivery was only partial for the Aß4-16/Ctr1-14 pair, in agreement with the higher complex stability for the former peptide. Moreover, the reaction was very slow and took ca. 40 h to reach equilibrium under the given experimental conditions. In either case of Cu(II) exchange, no intermediate (ternary) species were present in detectable amounts. In contrast, both Aß species released Cu(I) to Ctr1-14 rapidly and in a quantitative fashion, but ternary intermediate species were detected in the analysis of XAS data. The results presented here are the first direct evidence of a Cu(I) and Cu(II) transfer between the human Ctr1 and Aß model peptides. These results are discussed in terms of the fundamental difference between the peptides' Cu(II) complexes (pleiotropic ensemble of open structures of Aß1-16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aß4-16) and the similarity of their Cu(I) complexes (both anchored at the tandem His13/His14, bis-His motif). These results indicate that Cu(I) may be more feasible than Cu(II) as the cargo for copper clearance from the synaptic cleft by Aß peptides and its delivery to Ctr1. The arguments in favor of Cu(I) include the fact that cellular Cu export and uptake proteins (ATPase7A/B and Ctr1, respectively) specifically transport Cu(I), the abundance of extracellular ascorbate reducing agent in the brain, and evidence of a potential associative (hand-off) mechanism of Cu(I) transfer that may mirror the mechanisms of intracellular Cu chaperone proteins.