No abstract

Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

T-705, a substituted pyrazine compound, has been found to exhibit potent anti-influenza virus activity in vitro and in vivo. In a time-of-addition study, it was indicated that T-705 targeted an early to middle stage of the viral replication cycle but had no effect on the adsorption or release stage. The anti-influenza virus activity of T-705 was attenuated by addition of purines and purine nucleosides, including adenosine, guanosine, inosine, and hypoxanthine, whereas pyrimidines did not affect its activity. T-705-4-ribofuranosyl-5-triphosphate (T-705RTP) and T-705-4-ribofuranosyl-5-monophosphate (T-705RMP) were detected in MDCK cells treated with T-705. T-705RTP inhibited influenza virus RNA polymerase activity in a dose-dependent and a GTP-competitive manner. Unlike ribavirin, T-705 did not have an influence on cellular DNA or RNA synthesis. Inhibition of cellular IMP dehydrogenase by T-705RMP was about 150-fold weaker than that by ribavirin monophosphate, indicating the specificity of the anti-influenza virus activity and lower level of cytotoxicity of T-705. These results suggest that T-705RTP, which is generated in infected cells, may function as a specific inhibitor of influenza virus RNA polymerase and contributes to the selective anti-influenza virus activity of T-705.Influenza is responsible for much morbidity and mortality in the world (7), and effective treatment is required. We now have two classes of drugs for the treatment of influenza, the ion channel blockers and the neuraminidase inhibitors. The ion channel blockers amantadine and rimantadine are of limited use because of a lack of activity against influenza B virus, side effects, and the rapid emergence of resistant virus strains (9). Neuraminidase inhibitors are effective against both influenza A and B viruses, and their usefulness in clinical treatment has been reported (2, 11). Ribavirin is a guanosine analogue and inhibits various RNA and DNA viruses, including influenza viruses (19). Its clinical efficacy against influenza virus after aerosol treatment of infected individuals has been reported (5), but it has been approved for use in only a few countries. Influenza viruses are able to undergo rapid antigenic changes, especially in the surface glycoproteins, and novel influenza virus variants which have high levels of virulence may emerge in the human population and cause severe disease. Therefore, exploration for novel anti-influenza virus agents is of most importance.T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has been found to have potent inhibitory activity against RNA viruses in vitro, especially influenza A, B, and C viruses. The selectivity index (the ratio of the 50% cell-inhibitory concentration [CC 50 ]/50% influenza virus-inhibitory concentration [IC 50 ]) was more than 6,000 (Table 1) (6). T-705 showed therapeutic efficacy in mouse infection models and had a profile different from those of ribavirin and oseltamivir (16).The present study describes an experimental approach to clarifying the mode of action of T-705, and the ...

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has been found to have potent and selective inhibitory activity against influenza virus. In an in vitro plaque reduction assay, T-705 showed potent inhibitory activity against influenza A, B, and C viruses, with 50% inhibitory concentrations (IC 50 s) of 0.013 to 0.48 g/ml, while it showed no cytotoxicity at concentrations up to 1,000 g/ml in Madin-Darby canine kidney cells. The selectivity index for influenza virus was more than 2,000. It was also active against a neuraminidase inhibitorresistant virus and some amantadine-resistant viruses. T-705 showed weak activity against non-influenza virus RNA viruses, with the IC 50 s being higher for non-influenza virus RNA viruses than for influenza virus, and it had no activity against DNA viruses. Orally administered T-705 at 100 mg/kg of body weight/day (four times a day) for 5 days significantly reduced the mean pulmonary virus yields and the rate of mortality in mice infected with influenza virus A/PR/8/34 (3 ؋ 10 2 PFU). These results suggest that T-705 may be a compound that is useful and highly selective against influenza virus infections and that has a mode of action different from those of commercially available drugs, such as amantadine, rimantadine, and neuraminidase inhibitors.

Bacteria release membrane vesicles (MVs) that play important roles in various biological processes. However, the mechanisms of MV formation in Gram-positive bacteria are unclear, as these cells possess a single cytoplasmic membrane that is surrounded by a thick cell wall. Here we use live cell imaging and electron cryo-tomography to describe a mechanism for MV formation in Bacillus subtilis. We show that the expression of a prophage-encoded endolysin in a sub-population of cells generates holes in the peptidoglycan cell wall. Through these openings, cytoplasmic membrane material protrudes into the extracellular space and is released as MVs. Due to the loss of membrane integrity, the induced cells eventually die. The vesicle-producing cells induce MV formation in neighboring cells by the enzymatic action of the released endolysin. Our results support the idea that endolysins may be important for MV formation in bacteria, and this mechanism may potentially be useful for the production of MVs for applications in biomedicine and nanotechnology.