Maturation of dengue virus nonstructural protein 4B in monocytes enhances production of dengue hemorrhagic fever-associated chemokines and cytokines.

High levels of viremia and chemokines and cytokines underlie the progression of severe dengue disease. Dengue virus (DENV) preferentially infects peripheral blood monocytes, which secrete elevated levels of immunomediators in patients with severe disease. Further, DENV nonstructural proteins (NS) are capable of modifying intracellular signaling, including interferon inhibition. We demonstrate that peak secretions of immunomediators such as IL-6, IL-8, IP-10, TNFα or IFNγ in DENV-infected monocytes correlate with maximum virus production and NS4B and NS5 are primarily responsible for the induction of immunomediators. Furthermore, we demonstrate that sequential NS4AB processing initiated by the viral protease NS2B3(pro) and via the intermediate 2KNS4B significantly enhances immunomediator induction. While the 2K-signal peptide is not essential for immunomediator induction, it plays a synergistic role with NS4B. These data suggest that NS4B maturation is important during innate immune signaling in DENV-infected monocytes. Given similar NS4B topologies and polyprotein processing across flaviviruses, NS4B may be an attractive target for developing Flavivirus-wide therapeutic interventions.

Rational design leads to more potent RNA interference against hepatitis B virus: factors effecting silencing efficiency.

RNA interference (RNAi) can be an effective antiviral agent; however, overexpression of RNAi can be toxic through competition with the endogenous microRNA (miRNA) machinery. We used rational design to identify highly potent RNAi that is effective at nontoxic doses. A statistical analysis was conducted to pinpoint thermodynamic characteristics correlated with activity. Sequences were selected that conformed to a consensus internal stability profile (ISP) associated with active RNAi, and RNAi triggers were expressed in the context of an endogenous miRNA. These approaches yielded highly active hepatitis B virus (HBV) RNAi. A statistical analysis found a correlation between activity and nucleation by binding within the seed sequence to accessible regions in the target RNA. Guide strands were selected for favorable strand biasing, but increased strand biasing did not correlate with potency, suggesting a threshold effect. Exogenous short hairpin RNAs (shRNAs), but not miRNAs were previously reported to compete with miRNAs for the miRNA/RNAi machinery. In contrast, we show that exogenous Polymerase III- but not Polymerase II-driven miRNAs compete with exogenous miRNAs, at multiple steps in the miRNA pathway. Exogenous miRNAs also compete with endogenous miR-21. Thus, competition with endogenous miRNAs should be monitored even when using miRNA-based therapeutics. However, potent silencing was achieved at doses where competition was not observed.

Generation of virulence factor variants in Staphylococcus aureus biofilms.

Several serious diseases are caused by biofilm-associated Staphylococcus aureus. Colonial variants occur in biofilms of other bacterial species, and S. aureus variants are frequently isolated from biofilm-associated infections. Thus, we studied the generation of variants with altered expression of virulence factors in S. aureus biofilms. We observed that the number of variants found in biofilms, as measured by hemolytic activity, varied for different strains. Further study of hemolytic activity and signaling by the accessory gene regulator (Agr) quorum-sensing system in one S. aureus strain revealed three primary biofilm subpopulations: nonhemolytic (Agr deficient), hemolytic (Agr positive), and hyperhemolytic (also Agr positive). The nonhemolytic variant became the numerically dominant subpopulation in the biofilm. The nonhemolytic variant phenotype was stable and heritable, indicating a genetic perturbation, whereas the hyperhemolytic phenotype was unstable, suggesting a phase variation. Transcription profiling revealed that expression of the agr locus and many extracellular virulence factors was repressed in the nonhemolytic variant. Expression of the agr-activating gene, sarU, was also repressed in the nonhemolytic variant, suggesting one potential regulatory pathway responsible for the Agr-deficient phenotype. We suggest that the development of these variants in biofilms may have important clinical implications.

Delays in Pseudomonas aeruginosa quorum-controlled gene expression are conditional.

Acyl-homoserine lactone (acyl-HSL) signaling is thought to mediate quorum sensing in many species of Proteobacteria. The opportunistic human pathogen Pseudomonas aeruginosa uses acyl-HSLs to regulate hundreds of genes, including many that code for extracellular virulence factors. The idea that the P. aeruginosa acyl-HSLs serve as quorum-sensing signals has been questioned recently because microarray experiments show that the addition of signals to cultures of P. aeruginosa does not advance the onset of transcription for most acyl-HSL-dependent genes. We show that, under specific conditions, the expression of many acyl-HSL-dependent genes can be triggered at low culture density by signal addition. If complex medium is conditioned by growth of a non-acyl-HSL-producing P. aeruginosa, signals can eliminate the delay in expression of a battery of acyl-HSL-dependent genes. Furthermore, for one representative gene, lasB, there is no delay when signals are added to P. aeruginosa growing in conditioned complex medium or in minimal medium. We conclude that complex medium contains an inhibitor or inhibitors that can prevent induction of many, but not all, acyl-HSL-regulated genes and that the inhibitor is consumed by P. aeruginosa. Our results show that acyl-HSL signals can trigger expression of a large number of acyl-HSL-dependent genes regardless of growth phase. In this way, signaling in P. aeruginosa appears similar to quorum signaling in other Proteobacteria.

Quorum sensing in Staphylococcus aureus biofilms.

Several serious diseases are caused by biofilm-associated Staphylococcus aureus, infections in which the accessory gene regulator (agr) quorum-sensing system is thought to play an important role. We studied the contribution of agr to biofilm development, and we examined agr-dependent transcription in biofilms. Under some conditions, disruption of agr expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation. Under those conditions where agr expression enhanced biofilm formation, biofilms of an agr signaling mutant were particularly sensitive to rifampin but not to oxacillin. Time lapse confocal scanning laser microscopy showed that, similar to the expression of an agr-independent fluorescent reporter, biofilm expression of an agr-dependent reporter was in patches within cell clusters and oscillated with time. In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm. Our studies indicate that the role of agr expression in biofilm development and behavior depends on environmental conditions. We also suggest that detachment of cells expressing agr from biofilms may have important clinical implications.