Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.

No abstract

Streptococcus agalactiae (Group B Streptococcus, GBS) is an encapsulated, Gram-positive bacterium that is a leading cause of neonatal pneumonia, sepsis and meningitis, and an emerging aquaculture pathogen. The zebrafish (Danio rerio) is a genetically tractable model vertebrate that has been used to analyze the pathogenesis of both aquatic and human bacterial pathogens. We have developed a larval zebrafish model of GBS infection to study bacterial and host factors that contribute to disease progression. GBS infection resulted in dose dependent larval death, and GBS serotype III, ST-17 strain was observed as the most virulent. Virulence was dependent on the presence of the GBS capsule, surface anchored lipoteichoic acid (LTA) and toxin production, as infection with GBS mutants lacking these factors resulted in little to no mortality. Additionally, interleukin-1β il1b and CXCL-8 (cxcl8a) were significantly induced following GBS infection compared to controls. We also visualized GBS outside the brain vasculature, suggesting GBS penetration into the brain during the course of infection. Our data demonstrate that zebrafish larvae are a valuable model organism to study GBS pathogenesis.

No abstract

Background Multidrug-resistant (MDR) Acinetobacter baumannii infections have high mortality rates and few treatment options. Synergistic drug combinations may improve clinical outcome and reduce further emergence of resistance in MDR pathogens. Here we show an unexpected potent synergy of two translation inhibitors against the pathogen: commonly prescribed macrolide antibiotic azithromycin (AZM), widely ignored as a treatment alternative for invasive Gram-negative pathogens, and minocycline, among the current standard-of-care agents used for A. baumannii . Methods Media-dependent activities of AZM and MIN were evaluated in minimum inhibitory concentration assays and kinetic killing curves, alone or in combination, both in standard bacteriologic media (cation-adjusted Mueller-Hinton Broth) and more physiologic tissue culture media (RPMI), with variations of bicarbonate as a physiologic buffer. Synergy was calculated by fractional inhibitory concentration index (FICI). Therapeutic benefit of combining AZM and MIN was tested in a murine model of A. baumannii pneumonia. AZM + MIN synergism was probed mechanistically by bacterial cytological profiling (BCP), a quantitative fluorescence microscopy technique that identifies disrupted bacterial cellular pathways on a single cell level, and real-time kinetic measurement of translation inhibition via quantitative luminescence. AZM + MIN synergism was further evaluated vs. other contemporary high priority MDR bacterial pathogens. Findings Although two translation inhibitors are not expected to synergize, each drug complemented kinetic deficiencies of the other, speeding the initiation and extending the duration of translation inhibition as verified by FICI, BCP and kinetic luminescence markers. In an MDR A. baumannii pneumonia model, AZM + MIN combination therapy decreased lung bacterial burden and enhanced survival rates. Synergy between AZM and MIN was also detected vs. MDR strains of Gram-negative Klebsiella pneumoniae and Pseudomonas aeruginosa , and the leading Gram-positive pathogen methicillin-resistant Staphylococcus aureus . Interpretation As both agents are FDA approved with excellent safety profiles, clinical investigation of AZM and MIN combination regimens may immediately be contemplated for optimal treatment of A. baumannii and other MDR bacterial infections in humans. Fund National Institutes of Health U01 AI124326 (JP, GS, VN) and U54 HD090259 (GS, VN). IC was supported by the UCSD Research Training Program for Veterinarians T32 OD017863.

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/ emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. V C 2013 International Society for Advancement of Cytometry

The development of new therapies that can prevent recurrence and progression of nonmuscle invasive bladder cancer remains an unmet clinical need. The continued cost of monitoring and treatment of recurrent disease, along with its high prevalence and incidence rate, is a strain on healthcare economics worldwide. The current work describes the characterization and pharmacological evaluation of VAX-IP as a novel bacterial minicell-based biopharmaceutical agent undergoing development for the treatment of nonmuscle invasive bladder cancer and other oncology indications. VAX-IP minicells selectively target two oncology-associated integrin heterodimer subtypes to deliver a unique bacterial cytolysin protein toxin, perfringolysin O, specifically to cancer cells, rapidly killing integrin-expressing murine and human urothelial cell carcinoma cells with a unique tumorlytic mechanism. The in vivo pharmacological evaluation of VAX-IP minicells as a single agent administered intravesically in two clinically relevant variations of a syngeneic orthotopic model of superficial bladder cancer results in a significant survival advantage with 28.6% (P = 0.001) and 16.7% (P = 0.003) of animals surviving after early or late treatment initiation, respectively. The results of these preclinical studies warrant further nonclinical and eventual clinical investigation in underserved nonmuscle invasive bladder cancer patient populations where complete cures are achievable.

Honey is a traditional remedy for dyspepsia, and is still used for this by some medical practitioners although there is no rational basis for its use. The finding that Helicobacter pylori is probably the causative agent in many cases of dyspepsia has raised the possibility that the therapeutic action of honey may be due to its antibacterial properties. Consequently, the sensitivity of Helicobacter pylori to honey was tested, using isolates from biopsies of gastric ulcers. It was found that all five isolates tested were sensitive to a 20% (v/v) solution of manuka honey in an agar well diffusion assay, but none showed sensitivity to a 40% solution of a honey in which the antibacterial activity was due primarily to its content of hydrogen peroxide. Assessment of the minimum inhibitory concentration by inclusion of manuka honey in the agar showed that all seven isolates tested had visible growth over the incubation period of 72 h prevented completely by the presence of 5% (v/v) honey.