Persistence Dynamics of Antimicrobial-Resistant Neisseria in the Pharynx of Rhesus Macaques.

Pharyngeal infections by Neisseria gonorrhoeae are often asymptomatic, making them difficult to treat. However, in vivo animal modeling of human pharyngeal infections by pathogenic Neisseria species is challenging due to numerous host tropism barriers. We have relied on rhesus macaques to investigate pharyngeal persistence of naturally occurring Neisseria species in response to antibiotics. These species include Neisseria mucosa, Neisseria oralis, and a species unique to macaques. Four animals previously treated intramuscularly with the fluoroquinolone enrofloxacin for 2 weeks were monitored for persistence of their preexisting Neisseria populations for a period of 10 weeks. Enrofloxacin exposure did not eliminate preexisting flora from two of the four animals. Characterization of a collection of macaque Neisseria isolates supported the hypothesis that pharyngeal persistence was linked to reduced enrofloxacin susceptibility conferred by mutations in either gyrA or parC Interestingly, we observed a change in neisserial population dynamics for several weeks following enrofloxacin exposure. Enrofloxacin appeared to promote competition between strains for dominance in the pharyngeal niche. Specifically, following enrofloxacin treatment, strains bearing single gyrA mutations and low MICs persisted long-term. In contrast, strains with both gyrA and parC mutations and high MICs became culturally undetectable, consistent with the hypothesis that they were less fit. Our study has provided insight into pharyngeal persistence dynamics of Neisseria species bearing fluoroquinolone resistance determinants. The rhesus macaque provides a valuable host animal that may be used in the future to simulate treatment failures associated with the presence of antimicrobial-resistant Neisseria spp. in the human pharynx.

The conformational epitope for a new Aß42 protofibril-selective antibody partially overlaps with the peptide N-terminal region.

Aggregation and accumulation of amyloid-ß peptide (Aß) is a key component of Alzheimer's disease (AD). While monomeric Aß appears to be benign, oligomers adopt a biologically detrimental structure. These soluble structures can be detected in AD brain tissue by antibodies that demonstrate selectivity for aggregated Aß. Protofibrils are a subset of soluble oligomeric Aß species and are described as small (< 100 nm) curvilinear assemblies enriched in ß-sheet structure. Our own in vitro studies demonstrate that microglial cells are much more sensitive to soluble Aß42 protofibrils compared to Aß42 monomer or insoluble Aß42 fibrils. Protofibrils interact with microglia, trigger Toll-like receptor signaling, elicit cytokine transcription and expression, and are rapidly taken up by the cells. Because of the importance of this Aß species, we sought to develop an antibody that selectively recognizes protofibrils over other Aß species. Immunization of rabbits with isolated Aß42 protofibrils generated a high-titer anti serum with a strong affinity for Aß42 protofibrils. The antiserum, termed AbSL, was selective for Aß42 protofibrils over Aß42 monomers and Aß42 fibrils. AbSL did not react with amyloid precursor protein and recognized distinct pathological features in AD transgenic mouse brain slices. Competition studies with an Aß antibody that targets residues 1-16 indicated that the conformational epitope for AbSL involved the N-terminal region of protofibrils in some manner. The newly developed antibody may have potential diagnostic and therapeutic uses in AD tissue and patients, and targeting of protofibrils in AD may have beneficial effects. Read the Editorial Highlight for this article on page 621. Cover Image for this issue: doi. 10.1111/jnc.13827.

Aß40 has a subtle effect on Aß42 protofibril formation, but to a lesser degree than Aß42 concentration, in Aß42/Aß40 mixtures.

Recent findings suggest that the senile plaques in Alzheimer's disease may contain soluble amyloid-ß peptide (Aß) fibril precursors along with insoluble fibrils. These soluble Aß species, including oligomers and protofibrils, have been well-studied in vitro and are formed via non-covalent self-assembly of Aß monomers. While both 40- and 42-residue forms of Aß are observed in the human body, the majority of the Aß aggregation work has been conducted on Aß42 or Aß40 separately, with relatively few investigations of mixtures. In order to study the effect of different combinations of Aß40 and Aß42 on protofibril formation, mixtures of either dry solid peptide, or purified Aß40 and Aß42 monomer solutions were mixed together and protofibril/monomer distributions were quantified. Increases in the Aß42/Aß40 ratio increased protofibril formation but the presence of Aß40 in the mixed Aß solutions had a significant negative impact on protofibril formation compared to equivalent solutions of pure Aß42. Protofibril size was less affected, but ß-sheet structure increased with protofibrils formed from higher Aß42/Aß40 ratio solutions. Direct measurement of Aß42/Aß40 ratios by C-terminal-selective ELISA found very little Aß40 incorporated into protofibrils. The cumulative data emphasizes the critical importance of Aß42, yet establishes Aß40 as a regulator of Aß42 aggregation.

Biophysical comparison of soluble amyloid-ß(1-42) protofibrils, oligomers, and protofilaments.

Some of the pathological hallmarks of the Alzheimer's disease brain are senile plaques composed of insoluble amyloid-ß protein (Aß) fibrils. However, much of the recent emphasis in research has been on soluble Aß aggregates in response to a growing body of evidence that shows that these species may be more neurotoxic than fibrils. Within this subset of soluble aggregated Aß are protofibrils and oligomers. Although each species has been widely investigated separately, few studies have directly compared and contrasted their physical properties. In this work, we examined well-recognized preparations of Aß(1-42) oligomers and protofibrils with multiangle (MALS) and dynamic (DLS) light scattering in line with, or following, size-exclusion chromatography (SEC). Multiple SEC-MALS analyses of protofibrils revealed molecular weight (Mw) gradients ranging from 200 to 2600 kDa. Oligomeric Aß species are generally considered to be a smaller and more nascent than protofibrils. However, oligomer Mw values ranged from 225 to 3000 kDa, larger than that for protofibrils. Root-mean-square radius (Rg) values correlated with the Mw trends with protofibril Rg values ranging from 16 to 35 nm, while oligomers produced one population at 40-43 nm with a more disperse population from 22 to 39 nm. Hydrodynamic radius (RH) measurements by DLS and thioflavin T fluorescence measurements indicated that protofibrils and oligomers had commonalities, yet electron microscopy revealed morphological differences between the two. SEC-purified Aß(1-42) monomer at lower concentrations was slower to nucleate but formed protofibrils (1500 kDa) or soluble protofilaments (3000 kDa) depending on the buffer type. The findings from these studies shed new light on the similarities and differences between distinct soluble aggregated Aß species.