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.