The diverse Fusobacterium genus contains species implicated in multiple clinical pathologies, including periodontal disease, preterm birth, and colorectal cancer. The lack of genetic tools for manipulating these organisms leaves us with little understanding of the genes responsible for adherence to and invasion of host cells. Actively invading Fusobacterium species can enter host cells independently, whereas passively invading species need additional factors, such as compromise of mucosal integrity or coinfection with other microbes. We applied whole-genome sequencing and comparative analysis to study the evolution of active and passive invasion strategies and to infer factors associated with active forms of host cell invasion. The evolution of active invasion appears to have followed an adaptive radiation in which two of the three fusobacterial lineages acquired new genes and underwent expansions of ancestral genes that enable active forms of host cell invasion. Compared to passive invaders, active invaders have much larger genomes, encode FadA-related adhesins, and possess twice as many genes encoding membrane-related proteins, including a large expansion of surface-associated proteins containing the MORN2 domain of unknown function. We predict a role for proteins containing MORN2 domains in adhesion and active invasion. In the largest and most comprehensive comparison of sequenced Fusobacterium species to date, we have generated a testable model for the molecular pathogenesis of Fusobacterium infection and illuminate new therapeutic or diagnostic strategies.

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

Spinal cord injury commonly leads to permanent motor and sensory deficits due to the limited regenerative capacity of the adult central nervous system (CNS). Nucleic acid-based therapy is a promising strategy to deliver bioactive molecules capable of promoting axonal regeneration. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to its cytotoxicity and low transfection efficiency in the presence of serum proteins. In this study, we synthesized cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP), by grafting low molecular weight PLGA (4kDa) to bPEI (25kDa) at approximately a 3:1 ratio as an efficient nonviral vector. We show that PgP micelle is capable of efficiently transfecting plasmid DNA (pDNA) and siRNA in the presence of 10% serum in neuroglioma (C6) cells, neuroblastoma (B35) cells, and primary E8 chick forebrain neurons (CFN) with pDNA transfection efficiencies of 58.8%, 75.1 %, and 8.1 %, respectively. We also show that PgP provides high-level transgene expression in the rat spinal cord in vivo that is substantially greater than that attained with bPEI. The combination of improved transfection and reduced cytotoxicity in vitro in the presence of serum and in vivo transfection of neural cells relative to conventional bPEI suggests that PgP may be a promising nonviral vector for therapeutic nucleic acid delivery for neural regeneration.

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Gram-negative bacteria produce outer membrane vesicles (OMVs) that play a critical role in cell-cell communication and virulence. Despite being isolated from a single population of bacteria, OMVs can exhibit heterogeneous size and toxin content, which can be obscured by assays that measure ensemble properties. To address this issue, we utilize fluorescence imaging of individual OMVs to reveal size-dependent toxin sorting. Our results showed that the oral bacterium Aggregatibacter actinomycetemcomitans (A.a.) produces OMVs with a bimodal size distribution, where larger OMVs were much more likely to possess leukotoxin (LtxA). Among the smallest OMVs (< 100 nm diameter), the fraction that are toxin positive ranges from 0-30%, while the largest OMVs (> 200 nm diameter) are between 70-100% toxin positive. Our single OMV imaging method provides a non-invasive way to observe OMV surface heterogeneity at the nanoscale level and determine size-based heterogeneities without the need for OMV fraction separation.

Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with periodontal disease tend to produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is secreted via a type 1 secretion system, from the bacterial cytosol across both the inner and outer membranes in a single step into the supernatant, where it is able to interact with host cells. Upon secretion, some of the toxin associates with the bacterial cell membrane, enabling its release in association with the surface of outer membrane vesicles (OMVs), small, spherical vesicles derived from the outer membrane. We have previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs, a highly abundant population of small (<100 nm in diameter) OMVs, and a less abundant population of large (>300 nm in diameter) OMVs. Here, we have investigated the association of LtxA with the two populations of OMVs varying in size. Our results indicate that surface-associated DNA drives the selective sorting of LtxA to large OMVs.

identified and analyzed by making meas urements on a population of single enti ties, for example, single liposomes [4][5][6][7][8][9] or EVs. [10,11] Traditional assays that treat a population as an ensemble obscure the distributions of particle properties, and they are blind to the asynchronous events that are revealed at the single cell, particle or molecule level.Single entity measurements can be obtained using microscopy, [12][13][14][15] spec troscopy, [16,17] flow cytometry, [10,18] mass spectrometry, [19,20] electrical, [21] electro chemical, [22] and separation methods, [23] all of which can reveal particle heteroge neities. However, many of these methods have weaknesses that limit their appli cability. For example, electron micro scopy has the requisite spatial resolution to observe individual small particles, but because of the required sample processing and fixing, temporal dynamics among particles cannot be determined. Flow cytometry is unable to characterize nanoscale (50-500 nm) objects, such as EVs, without special instrument modifications. Additionally, flow cytometry, capillary electrophoresis, and elec trochemical cytometry have highthroughput capabilities, but they are unable to analyze a single isolated particle over long periods of time, and specific individual particles are gener ally not recoverable for further analysis. Also, some of these methods destroy the particles as part of the measurement pro cess, which prevents multiplex analysis of individual particles. To overcome these drawbacks, new approaches are necessary that leverage the strengths of optical microscopy and enable multiplex highthroughput measurements on single biological particles to reveal their physical, chemical, and physiological heterogeneities.Chemicallyspecific capture of individual particles on sur faces can enable single particle analysis. [24,25] Furthermore, this approach can be improved by patterning the capture molecules in densely packed, highly ordered arrays, resulting in densely packed, highly ordered arrays of particles. [7] Pat terning the capture molecules on a surface results in the cap ture of thousands of particles in ordered arrays within a single image frame. Patterning capture molecules into highly ordered arrays ensures that neighboring particles are optically resolv able, while at the same time particle surface density is maxi mized. Additionally, capture molecule nanodots that are similar Analytical characterization of small biological particles, such as extracellular vesicles (EVs), is complicated by their extreme heterogeneity in size, lipid, membrane protein, and cargo composition. Analysis of individual particles is essential for illuminating particle property distributions that are obscured by ensemble measurements. To enable high-throughput analysis of individual particles, liftoff nanocontact printing (LNCP) is used to define hexagonal antibody and toxin arrays that have a 425 nm dot size, on average, and 700 nm periodicity. The LNCP process is rapid, simple, and does not require access ...

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