Small β‐amyloid (Aβ) 1–42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer’s disease (AD). Methods to reduce the level of Aβ, prevent Aβ aggregation, and eliminate existing Aβ aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Aβ oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Aβ42 oligomer. Circular dichroism spectroscopy reveals monomeric Aβ42 peptide remains as a random coil/α‐helix structure in the presence of CP2 over 48 h. Atomic force microscopy studies show CP2 exhibits similar ability to inhibit Aβ42 aggregation as that of Congo red and curcumin. Atomic force microscopy closed‐fluid cell study demonstrates that CP2 disaggregates Aβ42 oligomers and protofibrils. CP2 also blocks Aβ fibrillations using a protein quantification method. Treatment of 5× familial Alzheimer’s disease mice, a robust Aβ42‐producing animal model of AD, with a 2‐week course of CP2 resulted in 40% and 50% decreases in non‐fibrillar and fibrillar Aβ species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Aβ aggregation and disaggregating existing Aβ oligomers and protofibrils.

Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo. We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-␥) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB, can serve as a model for the rational design of attenuated vaccines.

Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS) induces macrophages to synthesize prostaglandin E2 (PGE2). Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial) that result in up-regulation of the PGE2 biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE2 synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE2 in primary macrophages, we infected cells with individual mutants from the closely related strain F. tularensis subspecies novicida U112 (U112) two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE2 by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI). Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE2. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE2. We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE2, while U112 pdpA::Tn does not grow yet does induce PGE2. We also found that U112 iglC::Tn neither grows nor induces PGE2. These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE2 synthesis. These mutants provide a critical entrée into the pathways used in the host for PGE2 induction.

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