Venezuelan equine encephalitis virus (VEEV) poses a major public health risk due to its amenability for use as a bioterrorism agent and its severe health consequences in humans. ML336 is a recently developed chemical inhibitor of VEEV, shown to effectively reduce VEEV infection in vitro and in vivo. However, its limited solubility and stability could hinder its clinical translation. To overcome these limitations, lipid-coated mesoporous silica nanoparticles (LC-MSNs) were employed. The large surface area of the MSN core promotes hydrophobic drug loading while the liposome coating retains the drug and enables enhanced circulation time and biocompatibility, providing an ideal ML336 delivery platform. LC-MSNs loaded 20 ± 3.4 μg ML336/mg LC-MSN and released 6.6 ± 1.3 μg/mg ML336 over 24 hours. ML336-loaded LC-MSNs significantly inhibited VEEV in vitro in a dose-dependent manner as compared to unloaded LC-MSNs controls. Moreover, cell-based studies suggested that additional release of ML336 occurs after endocytosis. In vivo safety studies were conducted in mice, and LC-MSNs were not toxic when dosed at 0.11 g LC-MSNs/kg/day for four days. ML336-loaded LC-MSNs showed significant reduction of brain viral titer in VEEV infected mice compared to PBS controls. Overall, these results highlight the utility of LC-MSNs as drug delivery vehicles to treat VEEV.

Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown and to the organism's potential as a bioterrorism agent. AlthoughF. tularensishas an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulentF. tularensisstrains to survive and replicate in the amoebaAcanthamoeba castellaniiwas explored. We observe thatA. castellaniitrophozoites rapidly encyst in response toF. tularensisinfection and that this rapid encystment phenotype is caused by factor(s) secreted by amoebae and/orF. tularensisinto the coculture medium. Further, our results indicate that in contrast to the live vaccine strain LVS, virulent strains ofF. tularensiscan survive inA. castellaniicysts for at least 3 weeks postinfection and that the induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenicF. tularensisstrains block lysosomal fusion inA. castellanii. Taken together, these data suggest that interactions betweenF. tularensisstrains and amoebae may play a role in the environmental persistence ofF. tularensis.

Francisella tularensis, the zoonotic cause of tularemia, can infect numerous mammals and other eukaryotes. Although studyingF. tularensispathogenesis is essential to comprehending disease, mammalian infection is just one step in the ecology ofFrancisellaspecies.F. tularensishas been isolated from aquatic environments and arthropod vectors, environments in which chitin could serve as a potential carbon source and as a surface for attachment and growth. We show thatF. tularensissubsp.novicidaforms biofilms during the colonization of chitin surfaces. The ability ofF. tularensisto persist using chitin as a sole carbon source is dependent on chitinases, since mutants lackingchiAorchiBare attenuated for chitin colonization and biofilm formation in the absence of exogenous sugar. A genetic screen for biofilm mutants identified the Sec translocon export pathway and 14 secreted proteins. We show that these genes are important for initial attachment during biofilm formation. We generated defined deletion mutants by targeting two chaperone genes (secB1andsecB2) involved in Sec-dependent secretion and four genes that encode putative secreted proteins. All of the mutants were deficient in attachment to polystyrene and chitin surfaces and for biofilm formation compared to wild-typeF. novicida. In contrast, mutations in the Sec translocon and secreted factors did not affect virulence. Our data suggest that biofilm formation byF. tularensispromotes persistence on chitin surfaces. Further study of the interaction ofF. tularensiswith the chitin microenvironment may provide insight into the environmental survival and transmission mechanisms of this pathogen.