The pathogenicity factor HrpF interacts with HrpA and HrpG to modulate type III secretion system (T3SS) function and t3ss expression in Pseudomonas syringae pv. averrhoi.

To ensure the optimal infectivity on contact with host cells, pathogenic Pseudomonas syringae has evolved a complex mechanism to control the expression and construction of the functional type III secretion system (T3SS) that serves as a dominant pathogenicity factor. In this study, we showed that the hrpF gene of P. syringae pv. averrhoi, which is located upstream of hrpG, encodes a T3SS-dependent secreted/translocated protein. Mutation of hrpF leads to the loss of bacterial ability on elicitation of disease symptoms in the host and a hypersensitive response in non-host plants, and the secretion or translocation of the tested T3SS substrates into the bacterial milieu or plant cells. Moreover, overexpression of hrpF in the wild-type results in delayed HR and reduced t3ss expression. The results of protein-protein interactions demonstrate that HrpF interacts directly with HrpG and HrpA in vitro and in vivo, and protein stability assays reveal that HrpF assists HrpA stability in the bacterial cytoplasm, which is reduced by a single amino acid substitution at the 67th lysine residue of HrpF with alanine. Taken together, the data presented here suggest that HrpF has two roles in the assembly of a functional T3SS: one by acting as a negative regulator, possibly involved in the HrpSVG regulation circuit via binding to HrpG, and the other by stabilizing HrpA in the bacterial cytoplasm via HrpF-HrpA interaction prior to the secretion and formation of Hrp pilus on the bacterial surface.

Bimodal effects of fluoxetine on cerebral nitrergic neurogenic vasodilation in porcine large cerebral arteries.

Fluoxetine-induced relaxation of the smooth muscle of small cerebral arteries is thought beneficial in treating mental disorders. The present study was designed to examine effect of fluoxetine on neurogenic nitrergic vasodilation in large cerebral arteries, using in vitro tissue myography, techniques of electrophysiology, calcium imaging and biochemistry. In isolated porcine endothelium-denuded basilar arteries in the presence of U-46619-induced active muscle tone, fluoxetine in low concentration (<0.03 µM) significantly enhanced nicotine- and choline-induced relaxations. The vasorelaxation, however, was blocked by higher concentration of fluoxetine (>0.3 µM) with maximum inhibition at 3 µM. At this concentration, fluoxetine did not affect the basal tone or vasorelaxations induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, fluoxetine exclusively blocked nicotine-induced inward currents and calcium influx in cultured neurons of rat superior cervical ganglion and Xenopus oocytes expressing human α7-, α3ß2-, or α4ß2-nicotinic acetylcholine receptors (nAChRs). In addition, fluoxetine at 0.03 µM and 3 µM significantly enhanced and blocked, respectively, nicotine-induced norepinephrine (NE) release from cerebral perivascular sympathetic nerves. These results indicate that fluoxetine via axo-axonal interaction mechanism exhibits bimodal effects on nAChR-mediated neurogenic nitrergic dilation of basilar arteries. Fluoxetine in high concentrations decreases while in low concentrations it increases neurogenic vasodilation. These results from in vitro experimentation suggest that optimal concentrations of fluoxetine which increase or minimally affect neurogenic vasodilation indicative of regional cerebral blood flow may be important consideration in treating mental disorders.

Optical tweezers based active microrheology of sodium polystyrene sulfonate (NaPSS).

We used oscillatory optical tweezers to investigate the microrheological properties of Sodium polystyrene sulfonate (NaPSS; Mw = 70 kDa) polymer solutions with different concentrations from 0.001 mM to 10 mM in terms of elastic modulus G'(ω) and loss modulus G"(ω) as a function of angular frequency (ω) in the range of 6 rad/s to 6000 rad/s. The viscoelastic properties (including zero-shear-rate viscosity, crossing frequency and transition frequency) as a function of polymer concentration, deduced from our primary data, reveal the subtle structural changes in the polymer solutions as the polymer concentration increases from dilute to semi-dilute regimes, passing through the critical micelle formation concentration and the polymer overlapping concentration. The experimental results are consistent with the Maxwell model in some regime, and with the Rouse model in other, indicating the transient network character and the micelles formation in different regimes.

Effects of galU mutation on Pseudomonas syringae-plant interactions.

Bacterial galU coding for a uridine diphosphate-glucose pyrophosphorylase plays an important role in carbohydrates biosynthesis, including synthesis of lipopolysaccharides (LPS), membrane-derived oligosaccharides, and capsular polysaccharides. In this study, we characterized the galU mutant of Pseudomonas syringae pv. syringae 61 (Psy61), a necrotizing plant pathogen whose pathogenicity depends on a functional type III secretion system (T3SS), and showed that the Psy61 galU mutant had reduced biofilm formation ability, was nonmotile, and had an assembled T3SS structure but failed to elicit hypersensitive response in resistant plants and necrotic lesions in susceptible plants. Moreover, the defective LPS and other pathogen-associated molecular patterns (PAMPs) on the surface of the Psy61 galU mutant were capable of inducing PAMP-triggered immunity, which severely compromised the ability of the Psy61 galU mutant to survive in planta. Our results demonstrated that the complete LPS protected plant-pathogenic bacteria from host innate immunity, similar to what was found in animal pathogens, prior to the translocation of T3S effectors and bacterial multiplication.