MicroRNAs (miRNAs) are a class of small (ϳ22-nucleotide) regulatory molecules that block translation or induce degradation of target mRNAs. These have been identified in a wide range of organisms, including viruses. In particular, the oncogenic gammaherpesviruses Kaposi's sarcoma herpesvirus and Epstein-Barr virus encode miRNAs that could potentially regulate either viral or host genes. To determine if Marek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, encodes miRNAs, we isolated small RNAs from MDV-infected chicken embryo fibroblasts (CEF) and used the 454 Life Sciences sequencing technology to obtain the sequences of 13,679 candidate host and viral small RNAs. Eight miRNAs were found, five of which flank the meq oncogene and three that map to the latency-associated transcript (LAT) region of the genome. The meq gene is unique to pathogenic serotypes of MDV and is transcriptionally active during latency and transformation, and the LAT region of the MDV genome is antisense to the immediate-early gene ICP4. Secondary structure analysis predicted that the regions flanking the miRNAs could form hairpin precursors. Northern blot analysis confirmed expression of all miRNAs in MDV-infected CEF, MDV-induced tumors, and MDV lymphoblastoid cell lines. We propose that the MDV miRNAs function to enable MDV pathogenesis and contribute to MDV-induced transformation of chicken T cells.

The outer membrane vesicles (OMVs) from smooth B. melitensis 16 M and a derived rough mutant, VTRM1 strain, were purified and characterized with respect to protein content and induction of immune responses in mice. Proteomic analysis showed 29 proteins present in OMVs from B. melitensis 16 M; some of them are well-known Brucella immunogens such as SOD, GroES, Omp31, Omp25, Omp19, bp26, and Omp16. OMVs from a rough VTRM1 induced significantly higher expression of IL-12, TNFα, and IFNγ genes in bone marrow dendritic cells than OMVs from smooth strain 16 M. Relative to saline control group, mice immunized intramuscularly with rough and smooth OMVs were protected from challenge with virulent strain B. melitensis 16 M just as well as the group immunized with live strain B. melitensis Rev1 (P < 0.005). Additionally, the levels of serum IgG2a increased in mice vaccinated with OMVs from rough strain VTRM1 consistent with the induction of cell-mediated immunity.

Treatment and eradication of intracellular pathogens such as Brucella is difficult because infections are localized within phagocytic cells and most antibiotics, although highly active in vitro, do not actively pass through cellular membranes. Thus, an optimum strategy to treat these infections should address targeting of active drugs to the intracellular compartment where the bacteria replicate, and should prolong the release of the antibiotics so that the number of doses and associated toxicity can be reduced. We incorporated streptomycin and doxycycline into macromolecular nanoplexes with anionic homo- and block copolymers via cooperative electrostatic interactions among the cationic drugs and anionic polymers. The approach enabled simultaneous binding of both antibiotics into the nanoplexes, and their use resulted in an improvement in performance as compared with the free drugs. Administration of two doses of the nanoplexes significantly reduced the Brucella melitensis load in the spleens and livers of infected BALB/c mice. The nanoplexes were more effective than free drugs in the spleens (0.72-log and 0.51-log reductions, respectively) and in the livers (0.79-log and 0.42-log reductions, respectively) of the infected mice. Further research regarding the design of optimum nanoplex structures will be directed towards alterations in both the core and the shell properties to investigate the effects of the rates and pathways of entry into immune cells where the brucellae replicate.

Magnetic Block Ionomer Clusters (MBIClusters) with hydrophilic ionic cores and nonionic coronas have been prepared that have ultrahigh transverse NMR relaxivities together with capacities for incorporating high concentrations of polar antibiotic payloads. Magnetite-polymer nanoparticles were assembled by adsorbing the polyacrylate block of an aminofunctional poly(ethylene oxide-b-acrylate) (H2N-PEO-b-PAA) copolymer onto magnetite nanoparticles. The PEO blocks extended into aqueous media to keep the nanoparticles dispersed. Amines at the tips of the H2N-PEO corona were then linked through reaction with a PEO diacrylate oligomer to yield MBIClusters where the metal oxide in the precursor nanoparticles were distinctly separated by the hydrophilic polymer. The intensity average spacing between the magnetite nanoparticles within the clusters was estimated to be ~50 nm. These MBIClusters with hydrophilic intra-cluster space had transverse relaxivities (r2’s) that increased from 190 to 604 s−1 mM Fe−1 measured at 1.4 T and 37 °C as their average sizes increased. The clusters were loaded with up to ~38 wt% of the multi-cationic drug gentamicin. MRI scans focused on the livers of mice demonstrated that these MBIClusters are sensitive contrast agents.

Magnetic block ionomer complexes (MBICs) containing a combination of therapeutic and imaging agents are of great interest for delivering drugs and tracking their biodistribution with magnetic resonance imaging. With an aim to develop nanocarriers with high drug loadings that integrate magnetic nanoparticles into one system, we herein report antibiotic-laden MBICs based on assembly of poly(ethylene oxide-b-acrylate) (PEO-b-PAA) ionomers with nanomagnetite and gentamicin. The polymer was bound to the magnetic nanoparticle surfaces via ligand adsorption of the PAA block, thereby creating a double corona structure with a nonionic PEO shell and an ionic region rich in PAA. The portion of carboxylates that were not bound to the magnetite provided binding sites for drug loading via ionic complexation. PEO was chosen as a block copolymer segment to improve biocompatibility and aid in dispersion through interparticle steric repulsion. Intensity average diameters increased from 34 to 62 nm upon adding the drug, suggesting that the particles formed small clusters. Zeta potentials decreased from approximately −40 without gentamicin to approximately −10 mV with the drug, indicating that the drug effectively localized the charges in the MBIC cores. Approximately 35 wt % of the encapsulated gentamicin was released under physiological conditions within 10 h, and this was followed by slower release of another 7% by 18 h. The solid magnetite core serves as a multifunctional substrate for block ionomers to stably adsorb, thus acting as a pseudo-crosslinking site in the complexes that enhances their stability. Complexes between PEO-b-PAA and gentamicin without magnetite instantaneously dissociate in saline buffer. When the same copolymer was adsorbed onto magnetite, subsequent complexation with gentamicin resulted in stable complexes that withstood media with physiological ionic strength.

Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar–phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 μM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 μM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.

Brucella abortus has been shown to produce two siderophores: 2,3-dihydroxybenzoic acid (2,3-DHBA) and brucebactin. Previous studies on Brucella have shown that 2,3-DHBA is associated with erythritol utilization and virulence in pregnant ruminants. The biosynthetic pathway and role of brucebactin are not known and the only gene shown to be involved so far is entF. Using cre-lox methodology, an entF mutant was created in wild-type B. abortus 2308. Compared with the wild-type strain, the ΔentF strain showed significant growth inhibition in iron minimal media that became exacerbated in the presence of an iron chelator. For the first time, we have demonstrated the death of the ΔentF strain under iron-limiting conditions in the presence of erythritol. Addition of FeCl(3) restored the growth of the ΔentF strain, suggesting a significant role in iron acquisition. Further, complementation of the ΔentF strain using a plasmid containing an entF gene suggested the absence of any polar effects. In contrast, there was no significant difference in survival and growth between the ΔentF and wild-type strains grown in the murine macrophage cell line J774A.1, suggesting that an alternate iron acquisition pathway is present in Brucella when grown intracellulary.

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